RNAs from pathogens inhibit plant immunity

ABSTRACT

The present invention relates to pathogen-resistant plants comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette. Methods of making and cultivating pathogen-resistant plants are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application No. 61/886,004, filed Oct. 2, 2013, the entire content of which is incorporated by reference herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. MCB-0642843, IOS-1257576 awarded by the National Science Foundation, a NIH grant (R01 GM093008). The government has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file -2149-1.TXT, created on Nov. 20, 2014, 225,280 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Botrytis cinerea is a fungal pathogen that infects almost all vegetable and fruit crops and annually causes $10-100 billion losses worldwide. With its broad host range, B. cinerea is a useful model for studying the pathogenicity of aggressive fungal pathogens. Many pathogens of plants and animals deliver effectors into host cells to suppress host immunity (H. Ashida et al., Curr. Opin. Microbiol. 14, 16 (2011); M. Rafiqi et al., Curr. Opin. Plant Biol. 15, 477 (2012); T. O. Bozkurt et al., Curr. Opin. Plant Biol. 15, 483 (2012); H. Hilbi, et al., Traffic 13, 1187 (2012)).

sRNAs induce gene silencing by binding to Argonaute (AGO) proteins and directing the RNA-induced silencing complex (RISC) to genes with complementary sequences. sRNAs from both plant and animal hosts have been recognized as regulators in host-microbial interaction (5-8). Although sRNAs are also present in various fungi and oomycetes, including many pathogens (9-14), it has not been clear whether they regulate host-pathogen interaction.

BRIEF SUMMARY OF THE INVENTION

The present application provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette.

In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) of the sRNA. In some embodiments, the STTM is engineered from primers (a forward primer and a reverse primer) listed in Table 2. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary to the sRNA.

The present application also provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is an sRNA-resistant target that encodes a protein that functions in plant immunity, wherein the promoter is heterologous to the polynucleotide. In some embodiments, a plant into which the expression cassette has been introduced has enhanced pathogen resistance compared to a control plant lacking the expression cassette.

In some embodiments, the polynucleotide is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to any of SEQ ID NOS:4-13. In some embodiments, the polynucleotide is an sRNA-resistant target encoding mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or tomato mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the polynucleotide is an sRNA-resistant target of a gene listed in FIG. 1, Table 1, or Table 3. In some embodiments, the polynucleotide is resistant to gene silencing by an sRNA listed in Table 1. In some embodiments, the polynucleotide is resistant to gene silencing by Bc-siR3.1, Bc-siR3.2, or Bc-siR5.

In some embodiments, the sRNA comprises a sequence listed in Table 1. In some embodiments, the sRNA comprises the sequence of Bc-siR3.1, Bc-siR3.2, or Bc-siR5.

In some embodiments, the pathogen is Botrytis. In some embodiments, the pathogen is Botrytis cines.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is pathogen inducible. In some embodiments, the promoter is induced upon infection by Botrytis. In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis BIK1 (SEQ ID NO:1), Arabidopsis PDF1.2 (SEQ ID NO:2), or tomato TPK1b (SEQ ID NO:3). In some embodiments, the promoter is stress-inducible. In some embodiments, the promoter is tissue-specific. In some embodiments, the promoter is specifically expressed in the epidermis. In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis ML1 (SEQ ID NO:14) or tomato ML1 (SEQ ID NO:15).

In another aspect, the present invention provides for expression cassettes comprising: a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette; or comprising a promoter operably linked a polynucleotide that is an sRNA-resistant target that encodes a protein that functions in plant immunity, wherein the promoter is heterologous to the polynucleotide. Isolated nucleic acids comprising said expression cassettes are also provided.

In still another aspect, the present invention provides for expression vectors comprising an expression cassette as described herein.

In another aspect, methods of making a pathogen-resistant plant are provided. In some embodiments, the method comprises:

-   -   introducing the nucleic acid comprising an expression cassette         as described herein into a plurality of plants; and     -   selecting a plant comprising the expression cassette

In yet another aspect, methods of cultivating a plurality of pathogen-resistant plants are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bc-sRNAs silence host target genes in both Arabidopsis and S. lycopersicum during B. cinerea infection. (A) Bc-siR3.1, Bc-siR3.2, and Bc-siR5 were expressed during infection of Arabidopsis as detected at 18, 24, 48, and 72 hpi and, (B) S. lycopersicum leaves at 18, 24, 32, 48 hpi by RT-PCR. Actin genes of B. cinerea, and Arabidopsis and S. lycopersicum were used as internal controls. Similar results were obtained from three biological replicates. (C) The Arabidopsis targets of Bc-siRNAs were suppressed at 24, 32, and 48 hpi of B. cinerea infection. PDF1.2, BIK1 and β-tubulin were used as controls. (D) The S. lycopersicum target gene MAPKKK4 was suppressed upon B. cinerea infection. Expression (C and D) was measured by quantitative RT (qRT)-PCR using actin as an internal control. Error bars indicate standard deviation of three technical replicates. Similar results were seen in three biological replicates. (E) Co-expression of Bc-siR3.2 or Bc-siR5 with their host targets (HA-tagged) in N. benthamiana revealed target silencing by Western blot analysis. Co-expression of AtmiR395 or target site-mutated versions of target genes was used as controls. (F) Expression of YFP-MPK2 or its synonymously mutated version (YFP-MPK2-m) after infection of B. cinerea was observed by confocal microscopy. Co-expression of YFP-MPK2 and Bc-siR3.2 was used as a control. (G) Expression of the YFP sensors carrying a Bc-siR3.2 target site of MPK2 or a Bc-siR3.2 target site-m was analyzed after infection of B. cinerea. Samples were examined at 24 hpi. Upper panel: YFP; bottom panel: YFP/bright field overlay; scale bars (F, G), 37.5 μm. Error bars indicate standard deviation of 20 images (F, G). The asterisk indicates significant difference (two-tail t-test; p<0.01). Similar results were obtained in three biological replicates in E-G.

FIG. 2. Bc-sRNAs trigger silencing of host targets that are involved in host immunity. (A) Expression of Bc-siR3.1, BcsiR3.2, or Bc-siR5 in transgenic Arabidopsis ectopically expressing Bc-siRNAs under the Cauliflower Mosaic Virus promoter 35S (Bc-sRNAox) was examined by Northern blot analysis. Highly expressed lines were selected for the following experiments. (B) Bc-sRNAox lines showed constitutive silencing of respective Bc-siRNA target genes measured by qRT-PCR. Two independent lines for each Bc-sRNAs were examined. Similar results were observed in two generations of the selected transgenic lines. (C) Bc-sRNAox plants exhibited enhanced disease susceptibility to B. cinerea compared to the wild type. (D) Loss-of-function mutants of Bc-siR3.2 and Bc-siR5 targets mpk1 mpk2 and wak displayed enhanced disease susceptibility. In all pathogen assays (C and D), lesion sizes were measured at 96 hpi. Error bars indicate the standard deviation of 20 leaves. (E) Biomass of B. cinerea was measured by qPCR at 96 hpi. Error bars indicate standard deviation of three technical replicates. For C, D and E, similar results were obtained from three biological repeats. (F) Virus-induced gene silencing (VIGS) of MAPKKK4 exhibited enhanced disease susceptibility to B. cinerea in S. lycopersicum (examined at 72 hpi) compared to control plants (TRV-RB). RB is a late-blight resistance gene that is not present in tomato. We chose to use a TRV vector with a fragment from a foreign gene as a control to eliminate the potential side effect of viral disease symptoms caused by TRV empty vector. Spray inoculation was used because silencing sectors are not uniform within the VIGS plants. Three sets of experiments with each of 6-10 plants for each construct were performed, and similar results were obtained. The asterisk indicates significant difference (two-tail t-test, p<0.01) in C-F.

FIG. 3. Bc-sRNAs hijack Arabidopsis AGO1 to suppress host immunity genes. (A) Loading of Bc-siR3.1, Bc-siR3.2 and Bc-siR5 into Arabidopsis AGO1 during infection was detected by AGO1-IP followed by RT-PCR. AGO1 from B. cinerea-infected leaves harvested at 24, 32 and 48 hpi was pulled down by AGO1 peptide antibody, and RNA was extracted from the AGO1-IP fraction. As a control, non-infected leaves mixed with B. cinerea mycelium (at least twice as much as that in B. cinerea-infected leaves at 48 hpi) were used to rule out any binding between AGO1 and Bc-sRNAs during the experimental procedures. Similar results were obtained from at least three biological repeats. (B) Arabidopsis ago1-27 exhibited reduced disease susceptibility to B. cinerea compared to the wild type. Lesion size of at least 20 leaves and fungal biomass were measured at 96 hpi. (C) Silencing of MPK2, MPK1, PRXIIF, and WAK during B. cinerea infection was abolished in ago1-27. (D) Arabidopsis dcl1-7 exhibited enhanced disease susceptibility to B. cinerea compared to the wild type. Similar results were obtained from three biological repeats (B-D). The asterisk indicates significant difference (two-tail t-test, p<0.01) in B, D.

FIG. 4. B. cinerea dcl1 dcl2 double mutant is compromised in virulence. (A) B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants were impaired in generating Bc-siR3.1, Bc-siR3.2, and Bc-siR5 as revealed by RT-PCR. B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants, produced much weaker disease symptoms than the wild type in Arabidopsis (B) and S. lycopersicum (C), as demonstrated by the lesion size measured of 20 leaves at 96 hpi and 48 hpi, respectively. Similar results were obtained from three biological repeats. (D) Expression of the sensor YFP-Bc-siR3.2 target site was silenced by wild type B. cinerea upon infection, but not by the dcl1 dcl2 mutant at 24 hpi (scale bar: 75 μm). Error bars indicate standard deviation of 20 images. Experiments were repeated two times with similar results. (E) B. cinerea dcl1 dcl2 mutant was compromised in suppression of MPK2, MPK1, PRXIIF in Arabidopsis, and MAPKKK4 in S. lycopersicum. Similar results were seen in two biological repeats. (F) Arabidopsis Bc-siR3.1ox and Bc-siR3.2ox lines were more susceptible to B. cinerea dcl1 dcl2 strain than Col-0 wild type. (G) Enhanced disease phenotype of dcl1 dcl2 infection was also observed on three TRV-MAPKKK4 silenced S. lycopersicum plants. Experiments in F and G were repeated three times with similar results. B. cinerea biomass was quantified at 96 hpi. The asterisk (in B, C, D, F, G) indicates significant difference (two-tail t-test; p<0.01).

FIG. 5. Genomic map and read distribution of Bc-SIR3 and Bc-SIR5 loci. The genomic regions of 60 nt up- and downstream of the Bc-sRNA of interest were included. Sequence reads of Bc-siR3 and Bc-siR5 in B. cinerea-infected Arabidopsis (0, 24, 48, 72 hpi), B. cinerea-infected S. lycopersicum (leaf/fruit 0, 24, 72 hpi), or in vitro culture B. cinerea sRNA libraries (conidiospores, mycelia, total biomass) (see, FIG. 15) are shown in three individual panels. Bc-siR3 and Bc-siR5 reads are in red. In vitro culture B. cinerea sRNA libraries did not show a clear peak for Bc-siR3.1 or Bc-siR3.2 compared to B. cinerea-infected Arabidopsis and S. lycopersicum libraries, indicating that those Bc-siRNAs were induced during infection. Similarly, Bc-SIR5 showed induction upon infection.

FIG. 6. (A) Target site and target site mutated versions of Bc-siRNA Arabidopsis target genes that were used in this study (SEQ ID NOS:16, 17-19, 17 and 20-23, respectively). (B) B. cinerea mycelium coincided with target gene suppression of YFP-MPK2 (center), but not YFP-MPK2-m (right) in N. benthamiana at 24 hpi; YFP-MPK2 without fungal infection was used as a control (left). Upper panel: YFP; bottom panel: YFP/bright field overlay; scale bar: 50 μm. (C) A schematic diagram of the YFP sensor carrying a Bc-siR3.2 target site.

FIG. 7. Isolation and characterization of Bc-siRNA target mutants and Bc-siRNAox lines. (A) Isolation of a loss-of function mutant line for WAK gene (At5g50290). Expression of WAK was completely knocked out in the T-DNA insertion line shown by RT-PCR. (B) Induction of BIK1 expression in response to B. cinerea infection was reduced in Bc-siR3.1ox and Bc-iR3.2ox lines, mpk1 mpk2, and wak mutant lines. Relative transcript levels of BIK1 were measured by real time RT-PCR. Error bars indicate standard deviation (SD) of three technical replicates. Similar results were obtained from two biological repeats.

FIG. 8. S. lycopersicum MAPKKK4 gene knockdown by TRV-induced gene silencing. (A) Expression of MAPKKK in S. lycopersicum TRV-MAPKKK4 silenced plants was measured by qRT-PCR using actin as an internal control. Error bars indicate SD of three technical replicates. Similar results were obtained from three biological repeats. (B) TRV-MAPKKK4 silenced plants exhibited a dwarf phenotype as compared with control plants (TRV-RB).

FIG. 9. Bc-siR3.1 and Bc-siR5 were specifically loaded into Arabidopsis AGO1 during infection, but not into AGO2 or AGO4, as revealed by AGO-IP followed by RT-PCR. Endogenous plant sRNAs were used as internal controls for IP: At-miR398a for AGO1, At-miR393b* for AGO2, and At-siR1003 for AGO4.

FIG. 10. The sRNAs that have no predicted plant targets (Bc-siR394, Bc-siR233, Bc-siR269) or have predicted targets that were not down-regulated (Bc-siR9, Bc-siR24, Bc-siR67) by B. cinerea infection are not present in the AGO-associated fractions.

FIG. 11. Arabidopsis ago1-27 is more resistant to B. cinerea infection than wild-type. (A) ago1-27 displayed reduced disease phenotype upon B. cinerea infection. (B) Induction of BIK1 in response to B. cinerea infection was increased in ago1-27.

FIG. 12. The phylogenetic tree of DCL proteins in pathogenic fungi. Schizosaccharomyces pombe and Neurospora crassa were used as references. An oomycete pathogen Phytophthora infestans was also included.

FIG. 13. Generation of B. cinerea dcl1, dcl2 single mutants and the dcl1 dcl2 double mutant by homologous recombination. (A) Schematic diagram of Bc-DCL1 and Bc-DCL2 knockout strategy by homologous recombination. Black arrows indicate primers used for genotyping. (B) The dcl1, dcl2, and dcl1 dcl2 knockout strains were confirmed by RT-PCR. (C) B. cinerea dcl1, dcl2, and dcl1 dcl2 mutant strains showed gradual growth retardation and delayed development of conidiospores: upper panel shows radial growth after 3 days, bottom panel shows conidiation at 21 days. (D) Two Bc-sRNAs, Bc-microRNA-like RNA2 (Bc-milR2) and Bc-siR1498, were identified as Dicer-independent and were expressed in dcl1 dcl2.

FIG. 14. The biomass of the B. cinerea dcl1 dcl2 mutant strain was strongly reduced as compared with the wild-type strain during infection of both Arabidopsis (A) and S. lycopersicum (B), as quantified by qPCR at 72 hpi and 48 hpi, respectively.

FIG. 15. Statistical analysis of the sRNA libraries from cultured B. cinerea, B. cinerea-infected Arabidopsis, and B. cinerea-infected S. lycopersicum.

FIG. 16. The predicted host targets of sRNAs Bc-siR3.1, Bc-siR3.2, and Bc-siR5 (SEQ ID NOS:24, 25, 24, 26, 24, 27-31, 30, 32, 30, 33, 30, 34, 30, 35-37, 36, 38, 36, 39, 36, 40, 36, 41, 36 and 42, respectively). Normalized read counts are given in reads per million B. cinerea sRNAs. Reads were summed from individual sRNA libraries for each category: cultured B. cinerea, B. cinerea-infected Arabidopsis, B. cinerea-infected S. lycopersicum. Target gene alignment was scored as described in Materials and Methods.

DEFINITIONS

The term “pathogen-resistant” or “pathogen resistance” refers to an increase in the ability of a plant to prevent or resist pathogen infection or pathogen-induced symptoms. Pathogen resistance can be increased resistance relative to a particular pathogen species or genus (e.g., Botrytis), increased resistance to multiple pathogens, or increased resistance to all pathogens (e.g., systemic acquired resistance).

“Pathogens” include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif. (1988)). In some embodiments, the pathogen is a fungal pathogen. In some embodiments, the pathogen is Botrytis.

The term “plant immunity suppressing sRNA” refers to an sRNA that induces gene silencing in a plant of one or more genes that function or are predicted to function in plant immunity. For example, in some embodiments a plant immunity suppressing sRNA is an sRNA that induces gene silencing of a mitogen-activated protein kinase (e.g., MPK1, MPK2, or MAPKKK4), an oxidative stress-related gene (e.g., periredoxin (PRXIIF), or a cell wall-associated kinase (WAK). Exemplary plant immunity suppressing sRNAs are listed, for example, in FIG. 16 and Table 1.

The term “sRNA” refers to “small RNA,” a short non-coding RNA sequence. In some embodiments, an sRNA sequence comprises less than about 250 nucleotides (e.g., less than 250 nucleotides, less than 200 nucleotides, less than 150 nucleotides, less than 100 nucleotides, or less than 50 nucleotides). In some embodiments, an sRNA sequence comprises about 50-250 nucleotides, about 15-250 nucleotides, about 20-200 nucleotides, about 50-200 nucleotides, about 20-100 nucleotides, about 20-50 nucleotides, or about 20-30 nucleotides. In some embodiments, a sRNA sequence induces gene silencing, e.g., in a host plant. For example, in some embodiments a sRNA sequence induces gene silencing by directing a host's (e.g., host plant's) RNA-induced silencing complex (RISC) to genes with complementary sequences (“target genes”).

The term “sRNA-resistant target,” as used with reference to a polynucleotide sequence, refers to a polynucleotide sequence having a synonymous mutation relative to a sRNA target gene, wherein the polynucleotide sequence of the sRNA-resistant target comprises one or more nucleotide mutations relative to the polynucleotide sequence of the sRNA target gene that decreases the ability of the sRNA (e.g., a pathogen sRNA) to induce gene silencing of the sRNA-resistant target gene and wherein the amino acid sequence (e.g., protein sequence) that is encoded by the polynucleotide sequence of the sRNA-resistant target is identical to the amino acid sequence that is encoded by the polynucleotide sequence of the sRNA target gene. In some embodiments, the polynucleotide sequence of the sRNA-resistant target comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide mutations relative to the polynucleotide sequence of the sRNA target gene.

The term “nucleic acid” or “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

The phrase “nucleic acid encoding” or “polynucleotide encoding” refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The term “substantial identity” or “substantially identical,” as used in the context of polynucleotide or polypeptide sequences, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10⁻⁵, and most preferably less than about 10⁻²⁰.

The term “complementary to” is used herein to mean that a polynucleotide sequence is complementary to all or a portion of a reference polynucleotide sequence. In some embodiments, a polynucleotide sequence is complementary to at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, or more contiguous nucleotides of a reference polynucleotide sequence. In some embodiments, a polynucleotide sequence is “substantially complementary” to a reference polynucleotide sequence if at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the polynucleotide sequence is complementary to the reference polynucleotide sequence.

A polynucleotide sequence is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not or cannot be translated are expressly included by this definition. One of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially similar to a sequence of the gene from which it was derived.

The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types. An “inducible promoter” is one that initiates transcription only under particular environmental conditions or developmental conditions.

The term “plant” includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

As described in the Examples section below, it has been surprisingly discovered that small RNAs (sRNAs) from a plant pathogen can suppress genes involved in plant immunity. Without being bound to a particular theory, it is believed that the pathogen sRNAs suppress immunity in a host plant by using the host plant's own gene silencing mechanisms to suppress genes that function in plant immunity.

Thus, one aspect of the present invention relates to enhancing a plant's pathogen resistance by blocking, attenuating, or targeting for destruction the pathogen sRNAs. In some embodiments, a pathogen sRNA is blocked, attenuated, or targeted for destruction using a complementary polynucleotide sequence (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to the sRNA) or using a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the complementary polynucleotide sequence or STTM that targets the pathogen sRNA is expressed in a plant (e.g., in an expression cassette operably linked to a promoter), wherein the plant is less susceptible to the pathogen as compared to a control plant in which complementary polynucleotide sequence or STTM is not expressed.

In another aspect, the present invention relates to enhancing a plant's pathogen resistance by expressing sRNA-resistant target genes involved in plant immunity in plants to overcome the effect of the pathogen sRNAs. In some embodiments, the sRNA-resistant target genes are expressed under the control of a promoter (e.g., a pathogen-inducible promoter, a stress-inducible promoter, or a tissue-specific promoter).

II. Pathogen sRNAs and Attenuation of Pathogen sRNAs

In one aspect, methods of blocking or attenuating plant immunity-suppressing sRNAs of pathogens are provided. In some embodiments, the method comprises expressing in a plant a polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA. In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the method comprises expressing in the plant the polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA under the control of a promoter, e.g., a constitutively active promoter, an inducible promoter, or tissue-specific promoter (e.g., a stress inducible promoter, a pathogen inducible promoter, or an epidermis-specific promoter).

In another aspect, plants having blocked or attenuated function of pathogen sRNAs are provided. In some embodiments, the plant comprises a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, wherein the plant is less susceptible to the pathogen relative to a control plant lacking the expression cassette. In some embodiments, the expression cassette comprises a polynucleotide that encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the expression cassette comprises a polynucleotide that encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the expression cassette comprises a promoter that is an inducible promoter (e.g., stress inducible or pathogen inducible). In some embodiments, the expression cassette comprises a promoter that is a constitutively active promoter. In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific).

In yet another aspect, expression cassettes comprising a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the promoter is heterologous to the polynucleotide, or isolated nucleic acids comprising said expression cassettes, are provided. In some embodiments, the expression cassette comprises a polynucleotide that encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the expression cassette comprises a polynucleotide that encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the expression cassette comprises a promoter that is an inducible promoter (e.g., stress inducible or pathogen inducible). In some embodiments, the expression cassette comprises a promoter that is a constitutively active promoter. In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific). In some embodiments, a plant in which the expression cassette is introduced is less susceptible to the pathogen compared to a control plant lacking the expression cassette.

Pathogen sRNAs

In some embodiments, the plant immunity suppressing sRNA is from a viral, bacterial, fungal, nematode, or insect pathogen. In some embodiments, the sRNA is from a fungal pathogen. Examples of plant fungal pathogens include, but are not limited to, Botyritis, Magnaporthe, Sclerotinia, Puccinia, Fusarium, Mycosphaerella, Blumeria, Colletotrichum, Ustilago, and Melampsora. See, e.g., Dean et al., Mol Plant Pathol 13:804 (2012). In some embodiments, the pathogen is Botyritis. In some embodiments, the pathogen is Botyritis cines.

In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides, about 15-150 nucleotides, about 15-100 nucleotides, about 15-50 nucleotides, about 20-50 nucleotides, about 15-30, or about 20-30 nucleotides. In some embodiments, the pathogen sRNA comprises a sequence of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets (e.g., induces gene silencing of) a gene encoding a protein that functions or is predicted to function in plant immunity. In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets a gene that encodes mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets any of SEQ ID NOs:4-13 or a portion thereof.

In some embodiments, the pathogen sRNA comprises a sequence listed in FIG. 16 (e.g., Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001). In some embodiments, the pathogen sRNA comprises the sequence of Bc-siR3.1 (TTGTGGATCTTGTAGGTGGGC; SEQ ID NO:43), Bc-siR3.2 (TACATTGTGGATCTTGTAGGT; SEQ ID NO:44), or Bc-siR5 (TTTGACTCGGAATGTATACTT; SEQ ID NO:45).

Polynucleotides Targeting Pathogen sRNAs

In some embodiments, the function of a pathogen sRNA as described herein in a plant is blocked, attenuated, or reduced by expressing in the plant a polynucleotide that is complementary or substantially complementary to the sRNA or that mediates the destruction of the sRNA. As used herein, the term “mediates destruction of an sRNA” refers to inducing or promoting the degradation of a small RNA (e.g., by a small RNA degrading nuclease). In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) that targets the sRNA. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA.

Short Tandem Target Mimics

In some embodiments, a short tandem target mimic (STTM) construct is used to block or attenuate function or activity of the pathogen sRNA. STTMs are composed of two short polynucleotide sequences mimicking small RNA target sites (e.g., one or more pathogen sRNA sites as described herein), separated by a linker of an empirically determined optimal size. STTMs trigger efficient degradation of targeted sRNAs by small RNA degrading nucleases. See Yan et al., Plant Cell 24:415-427 (2012).

Typically, the STTM is designed to have two noncleavable sRNA binding sites separated by a spacer. The two noncleavable sRNA binding sites can be either identical (to target one specific sRNA) or slightly different to target two slightly different sRNAs. The optimal length of the spacer is typically from about 48 to 88 nucleotides, although shorter or longer spacer sequences can be used. The sequences of the spacer should be relatively AT rich and able to form a stable stem. Methods of designing and testing STTM constructs are described, e.g., in Yan et al., Plant Cell 24:415-427 (2012), and in Tang et al., Methods 58:118-125 (2012), incorporated by reference herein.

In some embodiments, the polynucleotide comprises an STTM construct that targets an sRNA sequence listed in FIG. 16 (e.g., Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001).

In some embodiments, the polynucleotide comprises an STTM construct that is generated using a pair of primers (a forward primer and a reverse primer) listed in Table 2. The STTM primers (e.g., the primers listed in Table 2) are used to amplify and clone into an expression vector a STTM construct having a sequence that targets an sRNA of interest (e.g., an sRNA listed in FIG. 16 or Table 1, e.g., any of Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001). In some embodiments, the STTM construct is expressed under the control of a promoter as described in Section IV below, e.g., a constitutively active promoter, an inducible promoter, or a tissue-specific promoter.

Antisense Technology

In some embodiments, antisense technology is used to block or attenuate function or activity of the pathogen sRNA. The antisense nucleic acid sequence that is transformed into plants is substantially identical to the pathogen sRNA sequence to be blocked. In some embodiments, the antisense polynucleotide sequence is complementary to the pathogen sRNA sequence to be blocked. However, the sequence does not have to be perfectly identical to inhibit expression. Thus, in some embodiments, an antisense polynucleotide sequence that is substantially complementary (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementary) to the pathogen sRNA sequence to be blocked can be used (e.g., in an expression cassette under the control of a heterologous promoter, which is then transformed into plants such that the antisense nucleic acid is produced). In some embodiments, the antisense polynucleotide is expressed under the control of a promoter as described in Section IV below, e.g., a constitutively active promoter, an inducible promoter, or a tissue-specific promoter.

In some embodiments, the polynucleotide encodes an antisense nucleic acid sequence that is complementary or substantially (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) complementary to an sRNA sequence listed in FIG. 16 (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001).

Other methods of using oligonucleotide or polynucleotide constructs for blocking the function of small RNAs as described herein can also be used, such as target mimicry (see, e.g., Franco-Zorrilla et al., Nat Genet. 39:1033-1037 (2007)) and “sponges” (see, e.g., Ebert et al., Nat. Methods 4:721-726 (2007)).

III. Expression of sRNA-Resistant Targets

In another aspect, methods of making plants that are resistant to one or more pathogen sRNAs are provided. In some embodiments, the method comprises:

-   -   introducing into a plant a heterologous expression cassette         comprising a promoter operably linked to a polynucleotide that         is an sRNA-resistant target that encodes a protein that         functions in plant immunity, wherein the promoter is         heterologous to the polynucleotide; and     -   selecting a plant comprising the expression cassette.

In another aspect, expression cassettes comprising a promoter operably linked to a polynucleotide encoding a sRNA-resistant target, isolated nucleic acids comprising said expression cassettes, or plants comprising said expression cassettes, are provided. In some embodiments, a plant into which the expression cassette has been introduced has enhanced pathogen resistance relative to a control plant lacking the expression cassette. In some embodiments, a plant into which the expression cassette has been introduced has enhanced resistance to a fungal pathogen (e.g., Botrytis, e.g., B. cinera) relative to a control plant lacking the expression cassette.

In some embodiments, the promoter is heterologous to the polynucleotide. In some embodiments, the polynucleotide encoding the sRNA-resistant target is operably linked to an inducible promoter. In some embodiments, the promoter is pathogen inducible (e.g., a Botrytis inducible promoter). In some embodiments, the promoter is stress inducible (e.g., an abiotic stress inducible promoter). In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific).

sRNA-Resistant Targets

In some embodiments, the polynucleotide is an sRNA-resistant target that encodes a protein that functions or is predicted to function in plant immunity. As used herein, an sRNA-resistant target is a polynucleotide sequence having a synonymous mutation of a sequence that is targeted by a pathogen sRNA. As used herein, the term “synonymous mutation” refers to a change, relative to a reference sequence, in a DNA sequence that encodes for a protein or peptide (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides relative to the reference sequence), wherein the change does not alter the amino acid that is encoded. For example, in some embodiments, pathogen sRNAs target plant immunity genes such as mitogen-activated protein kinases (including but not limited to, mitogen-activated protein kinase 1 (MPK1) or mitogen-activated protein kinase 2 (MPK2)); accordingly, in some embodiments an sRNA-resistant target comprises a synonymous mutation of a plant gene that encodes a mitogen-activated protein kinase (e.g., a synonymous mutation of MPK1 or MPK2).

In some embodiments, a polynucleotide sequence is an sRNA-resistant target if the polynucleotide sequence if the amino acid encoded by the polynucleotide sequence is produced at a detectable level. In some embodiments, a polynucleotide sequence is an sRNA-resistant target if the polynucleotide sequence if the amount of amino acid produced by a plant expressing the polynucleotide sequence in the presence of a pathogen sRNA is decreased by no more than 50%, 40%, 30%, 20%, 10%, 5%, or less relative to the amount of amino acid produced by a control plant expressing the polynucleotide sequence in the absence of the pathogen sRNA. Whether a polynucleotide is an sRNA-resistant target can be tested, for example, using a coexpression assay in Nicotiana benthamiana in which the sRNA is coexpressed with a polynucleotide sequence (e.g., a target gene or a synonymous mutation of the target gene) and the level of gene silencing induced by sRNA is measured. See, e.g., Example 1.

In some embodiments, the polynucleotide encodes a protein that functions or is predicted to function in plant immunity. In some embodiments, the polynucleotide comprises an sRNA-resistant target gene or predicted target gene listed in FIG. 16, Table 1, or Table 3. In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene that encodes mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene in tomato selected from Solyc08g081210.2.1, Solyc03g061650.1.1, Solyc01g108160.2.1, Solyc09g014790.2.1, Solyc03g112190.2.1, or Solyc07g066530.2.1. In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene in Vitis selected from VIT_10s0092g00240, VIT_12s0028g01140, VIT_06s0009g01890, VIT_10s0116g00190, VIT_05s0020g01790, VIT_01s0011g01000, VIT_05s0077g01510.

In some embodiments, the polynucleotide is substantially identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. In some embodiments, the polynucleotide is a homolog of any of SEQ ID NOS:4-13 (e.g., a homolog found in a species of Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, or Zea).

In some embodiments, the polynucleotide is substantially identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any of SEQ ID NOS:4-13, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide mutations relative to SEQ ID NOS:4-13, and encodes an identical protein as SEQ ID NOS:4-13. Non-limiting examples of nucleotide mutations (synonymous mutations) that can be made in the sequences of SEQ ID NOS:4-13 are described below in Example 3, as shown in the alignments of sRNA sequences to wild-type target gene sequences and mutated target gene sequences.

In some embodiments, the sRNA-resistant target gene comprises a polynucleotide sequence that is resistant to gene silencing by an sRNA listed in FIG. 16 or Table 1. In some embodiments, the sRNA-resistant target comprises a polynucleotide sequence that is resistant to gene silencing by Bc-siR3.1 (TTGTGGATCTTGTAGGTGGGC; SEQ ID NO:43), Bc-siR3.2 (TACATTGTGGATCTTGTAGGT; SEQ ID NO:44), or Bc-siR5 (TTTGACTCGGAATGTATACTT; SEQ ID NO:45).

IV. Polynucleotides and Recombinant Expression Vectors

The isolation of polynucleotides of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired polynucleotide in a cDNA or genomic DNA library from a desired plant species. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Alternatively, cDNA libraries from plants or plant parts (e.g., flowers) may be constructed.

The cDNA or genomic library can then be screened using a probe based upon a sequence disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide can be used to screen an mRNA expression library.

Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).

Polynucleotides can also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Once a polynucleotide sequence that is complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, or a polynucleotide that is a sRNA-resistant target, is obtained, it can be used to prepare an expression cassette for expression in a plant. In some embodiments, expression of the polynucleotide is directed by a heterologous promoter.

Any of a number of means well known in the art can be used to drive expression of the polynucleotide sequence of interest in plants. Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), epidermis, roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, expression can be conditioned to only occur under certain conditions (e.g., using an inducible promoter).

For example, a plant promoter fragment may be employed to direct expression of the polynucleotide sequence of interest in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.

Alternatively, the plant promoter may direct expression of the polynucleotide sequence of interest in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as the epidermis, leaves, or guard cells (including but not limited to those described in WO/2005/085449; U.S. Pat. No. 6,653,535; U.S. Pat. No. 7,834,243; EP Patent No. 1 888 754; Li et al., Sci China C Life Sci. 2005 April; 48(2):181-6; Husebye, et al., Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; Plesch, et al., Gene, Volume 249, Number 1, 16 May 2000, pp. 83-89(7), and Sessions et al., Plant J, October 1999, Vol. 20, pp. 259-263, each of which is incorporated by reference). Examples of environmental conditions that may affect transcription by inducible promoters include the presence of a pathogen, anaerobic conditions, elevated temperature, or the presence of light.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is stress inducible (e.g., inducible by abiotic stress). In some embodiments, the promoter is pathogen inducible. In some embodiments, the promoter is induced upon infection by Botrytis. Non-limiting examples of pathogen inducible promoters include Botrytis-Induced Kinase 1 (BIK1) and the plant defensing gene PDF1.2. See, e.g., Penninckx et al., Plant Cell 10:2103-2113 (1998); see also Veronese et al., Plant Cell 18:257-273 (2006). In some embodiments, the promoter is A. thaliana BIK1 (SEQ ID NO:1) or is substantially identical to A. thaliana BIK1 (SEQ ID NO:1). In some embodiments, the promoter is A. thaliana PDF1.2 (SEQ ID NO:2) or is substantially identical to A. thaliana PDF1.2 (SEQ ID NO:2). In some embodiments, the promoter is TPK1b (SEQ ID NO:3) or is substantially identical to TPK1b (SEQ ID NO:3).

In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is specifically expressed in the epidermis. Non-limiting examples of epidermis-specific promoters include Meristem Layer 1 (ML1). See, e.g., Takada et al., Development 140:1919-1923 (2013). In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis ML1 (SEQ ID NO:14) or tomato ML1 (SEQ ID NO:15).

In some embodiments, a polyadenylation region at the 3′-end of the coding region can be included. The polyadenylation region can be derived from a NH3 gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the sequences will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.

V. Production of Transgenic Plants

As detailed herein, embodiments of the present invention provide for transgenic plants comprising recombinant expression cassettes for expressing a polynucleotide sequence as described herein (e.g., a polynucleotide sequence that is complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, or a polynucleotide encoding a sRNA-resistant target). In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is derived from a species other than the species of the transgenic plant. It should be recognized that transgenic plants encompass the plant or plant cell in which the expression cassette is introduced as well as progeny of such plants or plant cells that contain the expression cassette, including the progeny that have the expression cassette stably integrated in a chromosome.

In some embodiments, the transgenic plants comprising recombinant expression cassettes for expressing a polynucleotide sequence as described herein have increased or enhanced pathogen resistance compared to a plant lacking the recombinant expression cassette, wherein the transgenic plants comprising recombinant expression cassettes for expressing the polynucleotide sequence have about the same growth as a plant lacking the recombinant expression cassette. Methods for determining increased pathogen resistance are described, e.g., in Section VI below.

A recombinant expression vector as described herein may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA construct can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA construct may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. While transient expression of the polynucleotide sequence of interest is encompassed by the invention, generally expression of construction of the invention will be from insertion of expression cassettes into the plant genome, e.g., such that at least some plant offspring also contain the integrated expression cassette.

Microinjection techniques are also useful for this purpose. These techniques are well known in the art and thoroughly described in the literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).

Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).

Transformed plant cells derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype such as enhanced pathogen resistance. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).

One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

The expression cassettes of the invention can be used to confer enhanced pathogen resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and Zea. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a vining plant, e.g., a species from the genus Vitis. In some embodiments, the plant is an ornamental plant. In some embodiments, the plant is a vegetable- or fruit-producing plant. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot.

VI. Selecting for Plants with Enhanced Pathogen Resistance

Plants with enhanced pathogen resistance can be selected in many ways. One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities. One method of selecting plants with enhanced pathogen resistance is to determine resistance of a plant to a specific plant pathogen. Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif.) (1988)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what pathogen, compound, or plant is used. Generally, enhanced resistance is measured by the reduction or elimination of disease symptoms (e.g., reduction in the number or size of lesions or reduction in the amount of fungal biomass on the plant or a part of the plant) when compared to a control plant. In some cases, however, enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. (1995) Science 268(5211): 661-7). Plants with enhanced pathogen resistance can produce an enhanced hypersensitive response relative to control plants.

Enhanced pathogen resistance can also be determined by measuring the increased expression of a gene operably linked a defense related promoter. Measurement of such expression can be measured by quantifying the accumulation of RNA or subsequent protein product (e.g., using northern or western blot techniques, respectively (see, e.g., Sambrook et al. and Ausubel et al.).

VII. Examples

The following examples are offered to illustrate, but not limit the claimed invention.

Example 1 Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways

Botrytis cinerea is a fungal pathogen that infects almost all vegetable and fruit crops and annually causes $10-100 billion losses worldwide. With its broad host range, B. cinerea is a useful model for studying the pathogenicity of aggressive fungal pathogens. Many pathogens of plants and animals deliver effectors into host cells to suppress host immunity (H. Ashida et al., Curr. Opin. Microbiol. 14, 16 (2011); M. Rafiqi et al., Curr. Opin. Plant Biol. 15, 477 (2012); T. O. Bozkurt et al., Curr. Opin. Plant Biol. 15, 483 (2012); H. Hilbi, et al., Traffic 13, 1187 (2012)). All the pathogen effectors studied so far are proteins. Here we find that small RNA (sRNA) molecules derived from B. cinerea can act as effectors to suppress host immunity.

sRNAs induce gene silencing by binding to Argonaute (AGO) proteins and directing the RNA-induced silencing complex (RISC) to genes with complementary sequences. sRNAs from both plant and animal hosts have been recognized as regulators in host-microbial interaction (5-8). Although sRNAs are also present in various fungi and oomycetes, including many pathogens (9-14), it has not been clear whether they regulate host-pathogen interaction.

To explore the role of B. cinerea sRNAs in pathogenicity, we profiled sRNA libraries prepared from B. cinerea (strain B05.10)-infected Arabidopsis thaliana Col-0 leaves collected at 0, 24, 48, and 72 h post inoculation (hpi) and from B. cinerea-infected Solanum lycopersicum (tomato) leaves and fruits at 0, 24, and 72 hpi. sRNA libraries prepared from B. cinerea mycelia, conidiospores and total biomass after 10 days of culture were used as controls. By using 100 normalized reads per million B. cinerea sRNA reads as a cutoff, we identified a total of 832 sRNAs that were present in both B. cinerea-infected Arabidopsis and S. lycopersicum libraries and had more reads in these two libraries than in the cultured B. cinerea libraries, with sequences exactly matching the B. cinerea B05.10 genome (15) but not Arabidopsis or S. lycopersicum genomes or cDNA (see, FIGS. 15 and 16 and Table 1). The closest sequence matches in Arabidopsis or S. lycopersicum contained a minimum of 2 mismatches. Among them, 27 had predicted microRNA-like precursor structures. A similar number of microRNA-like sRNAs was found in Sclerotinia sclerotiorum (9). We found that 73 Bc-sRNAs could target host genes in both Arabidopsis and S. lycopersicum under stringent target prediction criteria (FIG. 15). Among them, 52 were derived from 6 retrotransposon long terminal repeats (LTR) loci in the B. cinerea genome, 13 were from intergenic regions of 10 loci, and 8 were mapped to 5 protein coding genes.

Some of the predicted plant targets, such as MAPKs, are likely to function in plant immunity. To test whether Bc-sRNAs could indeed suppress host genes during infection, three Bc-sRNAs (Bc-siR3.1, Bc-siR3.2, and Bc-siR5) were selected for further characterization (FIG. 16). These Bc-sRNAs were among the most abundant sRNAs that were 21 nt in length and had potential targets likely to be involved in plant immunity in both Arabidopsis and S. lycopersicum. These sRNAs were also enriched after infection (FIGS. 1A-1B, FIG. 5, and FIG. 16), and were the major sRNA products from their encoding loci, LTR retrotransposons (FIG. 5). Bc-siR3.1 and Bc-siR3.2 were derived from the same locus with a four-nucleotide shift in sequence.

To determine whether Bc-sRNAs could trigger silencing of host genes, we examined the transcript levels of the predicted target genes after B. cinerea infection. The following Arabidopsis genes were targeted in the coding regions and were suppressed after B. cinerea infection: mitogen activated protein kinase 2 (MPK2) and MPK1, which are targeted by Bc-siR3.2; an oxidative stress-related gene peroxiredoxin (PRXIIF), which is targeted by Bc-siR3.1; and a putative cell wall-associated kinase gene (WAK), which is targeted by Bc-siR5 (FIG. 1C). In contrast, the plant defense marker genes PDF1.2 and BIK1 (P. Veronese et al., Plant Cell 18, 257 (2006)), which do not contain the Bc-sRNA target sites, were highly induced upon B. cinerea infection (FIG. 1C). We conclude that suppression of some but not all genes is a result of sequence-specific sRNA interaction and not due to cell death within infected lesions. Bc-siR3.2, which silences Arabidopsis MPK1 and MPK2, was enriched also in S. lycopersicum leaves upon B. cinerea infection and was predicted to target another member of the MAPK signaling cascade in S. lycopersicum, MAPKKK4 (FIG. 1B, FIG. 16). Expression of MAPKKK4 was indeed suppressed upon B. cinerea infection (FIG. 1D).

To confirm that the suppression of the targets was indeed triggered by Bc-sRNAs, we performed co-expression assays in Nicotiana benthamiana. Expression of HA-epitope tagged MPK2, MPK1, and WAK was reduced when they were co-expressed with the corresponding Bc-sRNAs but not when co-expressed with Arabidopsis miR395 that shared no sequence similarity (FIG. 1E). The silencing was abolished, however, when the target genes carried a synonymously mutated version of the relevant Bc-sRNA target sites (FIG. 6A, FIG. 1E). We also observed suppression of YFP-tagged target MPK2 by B. cinerea infection at 24 hpi (FIG. 1F and FIG. 6B); when the Bc-siR3.2 target site of MPK2 was mutated, infection by B. cinerea failed to suppress its expression (FIG. 1F). Thus, Bc-siR3.2 delivered from B. cinerea is sufficient for inducing silencing of wild type MPK2 but cannot silence target site-mutated MPK2. Similarly, of the YFP-sensors with wild type or mutated Bc-siR3.2 target sites (FIG. 6C), only the wild type sensor was suppressed after B. cinerea infection (FIG. 1G).

To test the effect of Bc-sRNAs on host plant immunity, we generated transgenic Arabidopsis plants that ectopically expressed Bc-siR3.1, Bc-siR3.2, or Bc-siR5 using a plant artificial miRNA vector (FIG. 2A) (17). These Bc-sRNA expression (Bc-sRNAox) lines showed normal morphology and development without pathogen challenge when compared to the wild type plants, and expression of the target genes was suppressed (FIG. 2B). With pathogen challenge, all of the Bc-sRNAox lines displayed enhanced susceptibility to B. cinerea (FIG. 2C, 2E). The results indicate that these Bc-sRNAs play a positive role in B. cinerea pathogenicity.

Enhanced disease susceptibility of the Bc-sRNAox lines suggests that the target genes of these Bc-sRNAs are likely to be involved in host immunity against B. cinerea. Plants with mutated target genes showed normal morphology and development without pathogen challenge. The Arabidopsis targets of Bc-siR3.2, MPK1 and MPK2, are homologs that share 87% amino acid identity. These genes are functionally redundant and are co-activated in response to various stress factors (18). The mpk1 mpk2 double mutant exhibited enhanced susceptibility to B. cinerea (FIG. 2D, 2E). A T-DNA knockout mutant of the Bc-siR5 target WAK (SALK_089827) (FIG. 7A) also displayed enhanced susceptibility to B. cinerea (FIG. 2D, 2E). Consistent with this, Bc-siRNAox lines as well as mpk1 mpk2 and wak showed lower induction of the defense marker gene BIK1 (FIG. 7B). These results suggest that the MPK1, MPK2, and WAK genes, all of which are targeted by Bc-sRNAs, participate in the plant's immune response to B. cinerea. To determine whether MAPKKK4 is involved in S. lycopersicum defense response against B. cinerea, we applied the virus-induced gene silencing (VIGS) approach to knock down MAPKKK4 in S. lycopersicum using tobacco rattle virus (TRV) (FIG. 8A) (19). VIGS of TRV-MAPKKK4 caused a dwarf phenotype (FIG. 8B). The MAPKKK4-silenced plants showed enhanced disease susceptibility in response to B. cinerea and contained >15 times more fungal biomass than the control plants (FIG. 2F). We conclude that Bc-sRNAs silence plant genes to suppress host immunity during early infection.

These fungal sRNAs hijack the plant's own gene silencing mechanism. 63 of the 73 Bc-sRNAs that had predicted Arabidopsis and S. lycopersicum targets were 20-22 nucleotides in length with a 5′ terminal U (see Table 1). This sRNA structure is favored for binding to AGO1 in Arabidopsis (S. J. Mi et al., Cell 133, 116 (2008); T. A. Montgomery et al., Cell 133, 128 (2008)). In order to determine whether Bc-sRNAs act through Arabidopsis AGO1, we immunoprecipitated AGO1 from B. cinerea-infected Arabidopsis collected at 24, 32 and 48 hpi and analyzed the AGO1-associated sRNAs. Bc-siR3.1, Bc-siR3.2 and Bc-siR5 were clearly detected in the AGO1-associated fraction pulled down from the infected plant samples but hardly in the control (FIG. 3A) or in the AGO2- and AGO4-associated sRNA fractions (FIG. 9). The sRNAs that had no predicted plant targets or had predicted targets that were not down-regulated by B. cinerea infection were not found in the AGO1-associated fractions (FIG. 10).

If AGO1 plays an essential role in Bc-sRNA-mediated host gene silencing, we would expect to see reduced disease susceptibility in the ago1 mutant since these Bc-sRNAs could no longer suppress host immunity genes. For plants carrying the ago1-27 mutant allele (J. B. Morel et al., Plant Cell 14, 629 (2002)) and were inoculated with B. cinerea, the disease level was significantly less than on the wild type (FIG. 3B and FIG. 11A). Consistent with this, BIK1 induction was increased compared to wild type (FIG. 11B). Furthermore, the expression of Bc-siR3.2 targets MPK2 and MPK1, Bc-siR3.1 target PRXIIF, and Bc-siR5 target WAK in ago1-27 was not suppressed compared to wild type infected plants after B. cinerea infection (FIG. 3C). On the contrary, Arabidopsis miRNA biogenesis mutant dicer-like (dcl) 1-7 that shows similar morphological defects to ago1-27 exhibited an enhanced disease level to B. cinerea (FIG. 3D). These results suggest that the increased resistance phenotype we observed in ago1-27 is not caused by any reduced vigor or pleiotropic phenotype, but due to the function of the Bc-siRNAs, and that Arabidopsis DCL1 is not required for the function of Bc-siRNAs. Thus, B. cinerea Bc-sRNAs evidently hijacked host RNAi machinery by loading into AGO1; the complex in turn suppressed host immunity genes.

To delete the siR3 and siR5 loci from the B. cinerea genome by homologous recombination would be an ideal way to confirm their function; however, it is not feasible because siR3 is from a LTR with 3 copies and siR5 is from a LTR with 13 copies. To better understand the function and biogenesis of the Bc-sRNAs, we chose to knock out the B. cinerea DCL genes, which encode the core sRNA processing enzymes. B. cinerea strain B05.10 possesses two Dicer-like genes (Bc-DCL1 and Bc-DCL2) (FIG. 12). We generated dcl1 and dcl2 single and dcl1 dcl2 double knockout mutant strains through homologous recombination (FIG. 13A-13B). We found that dcl1 and dcl2 single mutants showed reduced growth and delayed sporulation (FIG. 13C). The dcl1 dcl2 double mutant displayed a more obvious phenotype than each of the single mutants, suggesting partial functional redundancy between DCL1 and DCL2 in B. cinerea. Bc-siR3.1, Bc-siR3.2, and Bc-siR5 could not be detected in the dcl1 dcl2 double mutant (FIG. 4A), indicating that they were DCL-dependent, while two other Bc-siRNAs, Bc-milR2 and Bc-siR1498, could still be detected in dcl1 dcl2 double mutant (FIG. 13D). Fungi have diverse sRNA biogenesis pathways, and not all sRNAs are DCL-dependent (H. C. Lee et al., Mol. Cell 38, 803 (2010)). The dcl1 dcl2 double mutant caused significantly smaller lesions than the wild type or dcl1 and dcl2 single mutants on both Arabidopsis and S. lycopersicum leaves (FIG. 4B-4C), in consistence with the significantly reduced fungal biomass at 72 hpi in Arabidopsis and 48 hpi in S. lycopersicum (FIG. 14), which indicates that the virulence of the dell dcl2 mutant was greatly reduced. These results further support the conclusion that Bc-siRNAs, particularly Bc-siR3.1, Bc-siR3.2 and Bc-siR5 that depend on DCL function, contribute to the pathogenicity of B. cinerea. Mutation of dcl1 or dcl2 in B. cinerea caused delayed growth and sporulation (FIG. 13C) but had no effect on pathogenicity (FIG. 4B-4C). Furthermore, expression of the YFP sensor carrying the Bc-siR3.2 target site in N. benthamiana was silenced when infected with wild type B. cinerea. The suppression was abolished when inoculated with the dcl1 dcl2 strain (FIG. 4D), indicating that the dcl1 dcl2 double mutant was unable to generate Bc-siR3.2 to suppress the target. We also confirmed the inability of dcl1 dcl2 to suppress Bc-siR3.1 and Bc-siR3.2 target genes MPK2, MPK1, and PRXIIF in Arabidopsis and MAPKKK4 in tomato upon infection (FIG. 4E). Consistent with this, the dcl1 dcl2 virulence was partially restored when infected on Arabidopsis Bc-siR3.1ox and Bc-siR3.2ox plants as well as in tomato TRV-MAPKKK4 silenced plants (FIG. 4F-4G).

Animal and plant pathogens have evolved virulence or effector proteins to counteract host immune responses. Various protein effectors have been predicted or discovered in fungal or oomycete pathogens from whole-genome sequencing and secretome analysis (M. Rafiqi et al., Curr. Opin. Plant Biol. 15, 477 (2012); T. O. Bozkurt et al., Curr. Opin. Plant Biol. 15, 483 (2012)), although delivery mechanisms are still under active investigation (D. Kale et al., Cell 142, 284 (2010); S. Wawra et al., Curr. Opin. Microbiol. 15, 685 (2012); M. Rafiqi et al., Plant Cell 22, 2017 (2010); S. Schornack et al., Proc. Natl. Acad. Sci. USA 107, 17421 (2010); S. Wawra et al., Proc. Natl. Acad. Sci. USA 109, 2096 (2012)). Here, we show that sRNAs as well can act as effectors through a mechanism that silences host genes in order to debilitate plant immunity and achieve infection. The sRNAs from B. cinerea hijack the plant RNAi machinery by binding to AGO proteins which in turn direct host gene silencing. Another fungal plant pathogen, Verticllium (V.) dahliae, also depends on AGO1 function for its pathogenicity (U. Ellendorff, et al., J. Exp. Bot. 60, 591 (2009)). The implications of these findings suggest an extra mechanism underlying pathogenesis promoted by sophisticated pathogens with the capability to generate and deliver small regulatory RNAs into hosts to suppress host immunity.

Material and Methods

Generation of dcl1, dcl2 single and double mutants of B. cinerea

By using homologous recombination and the Agrobacterium tumefaciens-mediated transformation system adapted from Utermark and Karlovsky (U. Utermark, P. Karlovsky, Protocol Exchange, published online 20 Mar. 2008 (10.1038/nprot.2008.83)), we generated dcl1, dcl2 and dcl1 dcl2 deletion mutants in B. cinerea strain B05.10. Transformants were selected with 70 ppm hygromycin or 100 ppm NH⁴-glufosinate.

Plant Materials and Protocols

Plant materials used in this study are: Arabidopsis thaliana ecotype Col-0, Solanum lycopersicum (tomato) cultivar Moneymaker, and Nicotiana benthamiana, Arabidopsis knockout mutants mpk1 mpk2 (SALK_063847xSALK_019507) (D. Ortiz-Masia et al., FEBS Lett. 581, 1834-1840 (2007)) and wak (SALK_089827).

The Gateway pEarley vectors (with YFP & HA tags) were used for expression of Bc-sRNA target genes (K. W. Earley et al., Plant J. 45, 616-629 (2006)). Bc-sRNAs were cloned into the miRNA319a backbone vector (R. Schwab et al., Plant Cell 18, 1121-1133 (2006)) and transferred into the Gateway vector pEarley100 (without tag) for expression.

Transient co-expression assays in N. benthamiana were performed as described in (X. Zhang et al., Mol. Cell 42, 356-366 (2011)).

Virus-induced gene silencing (VIGS) was performed by cloning a 294-bp MPKKK4 gene fragment into the TRV2 vector (Y. L. Liu et al., Plant J. 31, 777-786 (2002)).

Pathogen Assay

Four-week-old plants were inoculated by applying a single 20 μl droplet per leaf or by spray-inoculating the entire plant, using 2×10⁵ spores/ml for Arabidopsis and 1×10⁴ spores/ml for S. lycopersicum and N. benthamiana. Disease was assessed by measuring lesion size (ImageJ software) and/or by quantifying B. cinerea biomass using quantitative PCR with B. cinerea-specific ITS primers (FIG. 8).

Confocal Microscopy

YFP-tagged protein expression in N. benthamiana was quantified using the confocal microscopy system Leica SP2. Z-series images (10 images in a distance of 0.7 μM) were merged to gain average signal intensity. Merged images were exported as TIFF files and YFP quantity was measured using the ImageJ software.

AGO Immunoprecipitation (IP)

Arabidopsis AGO IP (X. Zhang et al., Mol. Cell 42, 356-366 (2011)) was conducted with 5 g fresh leaves collected at 24, 32 and 48 h after spray inoculation with B. cinerea. Uninfected leaves mixed with at least double amount of B. cinerea biomass as in 48 hpi samples were used as a control. AGO1 was purified with a peptide-specific antibody. AGO2 and AGO4 IPs were conducted using native promoter-driven transgenic epitope HA-tagged and c-MYC-tagged lines, respectively and commercial HA and c-MYC antibodies.

sRNA RT-PCR

RNA was extracted from B. cinerea-infected plant tissue or the AGO pull-down fraction using the Trizol method. Purified RNA was treated with DNase I and then used in RT-PCR (E. Varkonyi-Gasic et al., Plant Methods 3, 12 (2007)) to detect Bc-sRNAs. 35-40 cycles were used for detecting Bc-sRNAs, 22-28 cycles were used for detecting actin genes from Arabidopsis, S. lycopersicum and B. cinerea. Primers used for reverse transcription and amplification of Bc-siRNAs are listed in Table 2.

sRNA cloning and Illumina HiSeq data analysis

sRNAs (18-28 nucleotides) were isolated by 15% PAGE and libraries were constructed using the miRCat cloning system and deep sequencing was performed on an Illumina HiSeq 2000. The sequence datasets of sRNA libraries from B. cinerea (GSE45320), B. cinerea-infected Arabidopsis (GSE45323) and B. cinerea-infected S. lycopersicum (GSE45321) are available at the NCBI database. The sRNA sequencing reads were preprocessed with the procedure of quality control and adapter trimming by using fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Following adapter trimming, sequences were mapped to B. cinerea B05.10, Arabidopsis (TAIR10), or S. lycopersicum (ITAG_SL2.40) genomes and only the reads that matched perfectly to each genome were used for further analysis. The read number for each distinct sRNA was normalized to the total B. cinerea mapped reads in B. cinerea-infected A. thaliana and S. lycopersicum libraries. The ratio of total B. cinerea mapped reads of A. thaliana and S. lycopersicum libraries is 2.5:1, so we divide the normalized siRNA read number of S. lycopersicum by 2.5.

The sRNAs we selected have satisfied the following conditions: 1) it must be present in both B. cinerea-infected A. thaliana and S. lycopersicum libraries; 2) its normalized read number was larger than 100 in A. thaliana or S. lycopersicum libraries; 3) its normalized reads must be higher than that in cultured B. cinerea libraries and 4) it has predicted targets in both A. thaliana and S. lycopersicum.

Target gene prediction for Bc-sRNA was performed using TAPIR1.1 (E. Bonnet et al., Bioinformatics 26, 1566-1568 (2010)) with more stringent requirement than described in (E. Bonnet et al., Bioinformatics 26, 1566-1568 (2010)). No gap or bulge within the alignment between the sRNA and the target was allowed, and the 10th nucleotide of the sRNA must perfectly match its target. At most one mismatch or two wobbles was allowed from position 2 to 12. A maximum of two continuous mismatches was allowed and a score of 4.5 was used as a cutoff. If a sRNA has predicted targets in both A. thaliana and S. lycopersicum, it was selected. The sRNAs were grouped if their 5′ end position and 3′ end position were within 3 nucleotides on the genomic loci. We presented the selected sRNAs with targets in both A. thaliana and S. lycopersicum in Table 1.

TABLE 1 Bc-sRNAs that have predicted targets in both Arabidopsis and S. lycopersicum. Bc-siRNA ID, locus, and siRNA sequence Normalized read counts   SEQ Target gene ID/ Putative function (5′-3′) A S B Target gene alignment and aligned score ID NO: AS* target site of target gene siR1 SIR1 LTR transposon TCGAAGCAAGAGTAGAATT 147.4 3015.92 36.4

46   47 4.5 AT5G06290.1 686~708(CDS) 2-cysteine peroxiredoxin B CTG (SEQ ID NO: 46)

46   48 4.25 Solyc01g068070.2.1 1754~1776 (cDNA) Wd-repeat protein (AHRD V1 *-*- C1FDE0_9CHLO);  contains Interpro domain(s) IPR017986 WD40 repeat, region siR1010 SIR1010 Intergenic region 2484.9 1644.16 2403.2

49   50 4.5 AT1G69330.1 566~587(CDS) RING/U-box superfamily protein TCGGGGGAATTTTT GATTGCT   (SEQ ID NO: 49)

49   51 4.5 Solyc07g018350.2.1 581~602(cDNA) DNA mismatch repair protein muts (AHRD V1 *-*- Q16P35_AEDAE); contains Interpro domain(s) IPR015536 DNA mismatch repair protein MutS-homolog MSH6 siR3.1 SIR2 LTR transposon TTGTGGATCTTGTA 812.1 1231.08 49.9

52   53 3.25 AT1G50760.1 86~107(CDS) Aminotransferase-like, plant mobile domain family protein GGTGGGC(SEQ ID NO: 52)

52   54 4.5 AT3G06050.1 333~354(CDS) peroxiredoxin IIF

52   55 4 AT5G46795.1 401~422(CDS) microspore-specific promoter 2

52   56 4.25 Solyc01g108160.2.1 3210~3231(cDNA) Autophagy-related  protein 2 (AHRD V1 *-*- C1GCV2_PARBD); contains Interpro domain(s) IPR015412 ATG2, C-terminal

52   57 4.5 Solyc09g014790.2.1 1194~1215(cDNA) Class E vacuolar protein-sorting machinery protein  hse1 (AHRD V1 *--- HSE1_EMENI); contains Interpro domain(s) IPR018205 VHS subgroup siR3.2 SIR2 LTR transposon TACATTGTGGATCT TGTAGGT 202.1 996.52 33.1

58   59 4.5 AT1G10210.1 291~312(CDS) mitogen-activated protein kinase 1 (SEQ ID NO: 58)

58   60 3 AT1G59580.1 353~374(CDS) mitogen-activated protein kinase  homolog 2

58   61 4 AT3G16830.1 585~606(CDS) TOPLESS-related 2

58   62 4.5 AT4G28300.1 1444~1465(CDS) Protein of unknown function (DUF1421)

58   63 3.5 Solyc03g061650.1.1 907~928(cDNA) F-box/LRR-repeat protein At3g26922 (AHRD V1 *-*- FBL47_ARATH);  contains Interpro domain(s) IPR006566 FBD-like

58   64 4.5 Solyc09g091030.2.1 1510~1531(cDNA) Beta-amylase (AHRD  V1 **** E0AE02_SOLLC); contains Interpro domain(s) IPR013781 Glycoside hydrolase, subgroup, catalytic core

65   66 4.5 Solyc08g081210.2.1 1936~1956(cDNA) MPKKK4 siR1008 SIR6 CDS (spurious gene) TGTGATGATGATCA GTTTATGC (SEQ ID 4255.7 635.28 299.8

67   68 4 AT1G04650.1 2418~2440(CDS) unknown protein, hypothetical protein NO: 67)

67   69 4 AT4G39180.2 1911~1933(3′UTR) Sec14p-like phosphatidylinositol transfer family protein

67   70 3.5 AT5G36940.1 221~243(CDS) cationic amino acid transporter 3

67   71 4.25 Solyc05g012030.1.1 603~625(cDNA) At1g69160/F4N2_9  (AHRD V1 ***- Q93Z37_ARATH)

67   72 4.5 Solyc06g076130.2.1 1605~1627(cDNA) Unknown Protein (AHRD V1) siR5 SIR3 LTR transposon TTTGACTCGGAAT GTATACTT (SEQ ID 1710 1380 302.6

73   74 4.5 AT3G05860.1 655~676(CDS) MADS-box transcription factor family protein NO: 73)

73   75 4 AT3G07730.1 491~512(CDS) unknown protein, hypothetical protein, uncharacterized  protein

73   76 4 AT3G08530.1 3491~3512(CDS) Clathrin, heavy chain

73   77 4.5 Solyc03g112190.2.1 1764~1785(cDNA) Pentatricopeptide repeat-containing protein (AHRD V1 ***- D7LRK9_ARALY); contains Interpro domain(s) IPR002885 Pentatricopeptide repeat

73   78 4 Solyc07g066530.2.1 910~931(cDNA) Mitochondrial import receptor subunit TOM34 (AHRD V1 *--- B5X380_SALSA); contains Interpro domain(s) IPR011990 Tetratricopeptide-like helical

79   80 4.25 AT5G50290 495~515(CDS) wall associated kinase siR9 SIR6 CDS (spurious gene) TTTTATGATGAGC ATTTTTAGA (SEQ ID 3847.8 120.16 231.7

81   82 4.5 AT1G73880.1 146~168(CDS) UDP-glucosyl  transferase 89B1 NO: 81)

81   83 4 Solyc04g005540.2.1 1920~1942(cDNA) Cc-nbs-lrr, resistance protein

81   84 4.25 Solyc05g007170.2.1 7265~7287(cDNA) Cc-nbs-lrr, resistance protein with an R1  specific domain

81   85 4 Solyc07g017880.2.1 780~802(cDNA) Peroxidase (AHRD V1 **** D4NYQ9_9ROSI); contains Interpro domain(s) IPR002016 Haemperoxidase, plant/fungal/bacterial

81   86 3.5 Solyc10g050580.1.1 306~328(cDNA) Protein binding protein (AHRD V1 ***- D7M3B0_ARALY)

81   87 4.5 Solyc11g013490.1.1 561~583(cDNA) Beta-1,3- galactosyltransferase 6 (AHRD V1 ***- B6UBH3_MAIZE);  contains Interpro domain(s) IPR002659 Glycosyl transferase, family 31 siR10 SIR2 LTR transposon TTTTCTAGGTTGTA GGGTGCT (SEQ ID 2234.2 689.6 56.5

88   89 4.25 AT1G63860.1 1124~1145(CDS) Disease resistance protein (TIR-NBS-LRR class) family NO: 88)

88   90 4 AT5G09260.1 511~532(CDS) vacuolar protein sorting-associated protein 20.2

88   91 4.5 Solyc04g050970.2.1 19~40(cDNA) Receptor protein kinase-like protein (AHRD V1 **** Q9LRY1_ARATH); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase

88   92 4.25 Solyc05g014650.2.1 541~562(cDNA) Iojap-like protein (AHRD V1 *-*- B5ZUF1_RHILW); contains Interpro domain(s) IPR004394 Iojap-related protein siR18 SIR1 LTR transposon TAGCCAAAACAG AGTCGATCA (SEQ ID 155.7 1260.68 16.2

93   94 4.5 AT2G01110.1 511~532(CDS) Sec-independent periplasmic protein translocase NO: 93)

93   95 4.5 AT2G31980.1 490~511(CDS) PHYTOCYSTATIN 2

93   96 4.5 AT3G26300.1 1345~1366(CDS) cytochrome P450, family 71, subfamily B, polypeptide 34

93   97 4.5 AT3G47440.1 366~387(CDS) tonoplast intrinsic protein 5;1

93   98 4.5 AT4G37160.1 52~73(CDS) SKU5 similar 15

93   99 4 Solyc02g071770.2.1 1000~1021(cDNA) DUF1264 domain protein (AHRD V1 **-- A1CBM4_ASPCL); contains Interpro domain(s) IPR010686 Protein of unknown function DUF1264

93   100 4 Solyc03g059420.2.1 2896~2917(cDNA) Sister chromatid cohesion 2 (AHRD V1 **-- D7M7D7_ARALY); contains Interpro domain(s) IPR016024 Armadillo-type fold

93   101 3.5 Solyc07g017240.1.1 1~22(cDNA) Unknown Protein  (AHRD V1) siR15 SIR3 LTR transposon TGTGTTGAACCTTG TTGTTTGA (SEQ ID 936.7 926.6 155

102   103 4.5 AT2G23080.1 1250~1272(3′UTR) Protein kinase superfamily protein NO: 102)

102   104 4 AT3G46920.1 3478~3500(CDS) Protein kinase superfamily protein with octicosapeptide/ Phox/Bem1p domain

102   105 3.5 AT5G48860.1 291~313(CDS) unknown protein, hypothetical protein, uncharacterized  protein

102   106 4.25 Solyc01g088020.2.1 786~808(cDNA) Protein transport protein sec31 (AHRD V1 **-- C8V1I6_EMENI); contains Interpro domain(s) IPR017986 WD40 repeat, region siR17 SIR6 CDS (spurious gene) TAAAATGATGAA TGGCACTGG (SEQ ID 1682.7 589.2 245.8

107   108 4.5 AT1G56190.1 1738~1759(3′UTR) Phosphoglycerate kinase family protein NO: 107)

107   109 4.5 AT1G72740.1 661~682(CDS) Homeodomain-like/ winged-helix DNA- binding family  protein

107   110 4.5 Solyc05g005950.2.1 262~283(cDNA) Solute carrier family 15 member 4 (AHRD V1 **-- S15A4_XENLA);  contains Interpro domain(s) IPR000109 TGF-beta receptor, type I/II extracellular region

107   111 4.5 Solyc05g005960.2.1 69~90(cDNA) Peptide transporter 1 (AHRD V1 **-* Q7XAC3_VICFA); contains Interpro domain(s) IPR000109 TGF-beta receptor, type I/II extracellular region

107   112 4 Solyc08g075450.2.1 222~243(cDNA) Nodulin-like protein (AHRD V1 ***- Q9FHJ9_ARATH); contains Interpro domain(s) IPR000620 Protein of unknown function DUF6, transmembrane

107   113 4 Solyc08g075460.2.1 424~445(cDNA) Nodulin-like protein (AHRD V1 ***- Q9FHJ9_ARATH); contains Interpro domain(s) IPR000620 Protein of unknown function DUF6, transmembrane siR22 SIR3 LTR transposon TAACGTGGTCAAG GGTGTAGT (SEQ ID 370 995.72 63.3

114   115 4.25 AT3G17360.1 625~646(CDS) phragmoplast orienting kinesin 1 NO: 114)

114   116 4.5 AT5G66510.1 438~459(CDS) gamma carbonic anhydrase 3

114   117 3.5 Solyc01g005240.2.1 1912~1933(cDNA) Aspartokinase (AHRD V1 ***- B9RGY9_RICCO);  contains Interpro domain(s) IPR001341 Aspartate kinase region siR24 SIR3 LTR transposon TGATTGGTCCTCTC TGTTTGAC (SEQ ID 1210.2 651.72 429.9

118   119 3.5 AT5G04990.1 1226~1248(CDS) SAD1/UNC-84 domain protein 1 NO: 118)

118   120 4 Solyc02g069090.2.1 2009~2031(cDNA) Cathepsin B (AHRD V1 ***- Q1HER6_NICBE); contains Interpro domain(s) IPR015643 Peptidase C1A, cathepsin B

118   121 4.5 Solyc03g007390.2.1 2085~2107(cDNA) Pentatricopeptide  repeat-containing protein (AHRD V1 ***- D7ML46_ARALY); contains Interpro domain(s) IPR002885 Pentatricopeptide repeat

118   122 4.5 Solyc03g097450.2.1 1351~1373(cDNA) SWI/SNF complex subunit SMARCC1 (AHRD V1 *--- SMRC1_HUMAN); contains Interpro domain(s) IPR007526 SWIRM

118   123 4.5 Solyc09g089970.1.1 287~309(cDNA) Unknown Protein (AHRD V1) siR25 SIR2 LTR transposon TAGTGAATCAAAT TTTGGTTTT (SEQ ID 2747.8 15.64 20.8

124   125 3.75 AT5G41250.1 1349~1371(CDS) Exostosin family protein NO: 124)

124   126 4 AT5G44030.1 3330~3352(3′UTR) cellulose synthase A4

124   127 4.5 Solyc01g044240.2.1 1312~1334(cDNA) Unknown Protein (AHRD V1)

124   128 3.5 Solyc12g005790.1.1 512~534(cDNA) Peroxidase 27 (AHRD V1 ***- D7LAI1_ARALY); contains Interpro domain(s) IPR002016 Haemperoxidase, plant/fungal/ bacterial siR1015 SIR1015 Intergenic region TTGATGGTTGTCTG ATCGGT (SEQ ID 1200.3 574.4 2304

129   130 4 AT2G45030.1 2328~2348(3′UTR) Translation elongation factor EFG/EF2 protein NO: 129)

129   131 4.5 AT5G02500.1 954~974(CDS) heat shock cognate protein 70-1

129   132 4.5 Solyc05g005180.2.1 437~457(cDNA) Naphthoate synthase (AHRD V1 ***- A8I2W2_CHLRE); contains Interpro domain(s) IPR010198 Naphthoate synthase

129   133 4.5 Solyc06g036150.1.1 564~584(cDNA) Unknown Protein (AHRD V1)

129   134 4.5 Solyc07g043250.1.1 116~136(cDNA) Unknown Protein (AHRD V1); contains Interpro domain(s) IPR008889 VQ

129   135 3.5 Solyc08g063100.1.1 438~458(cDNA) Ulp1 protease family C-terminal catalytic domain containing protein (AHRD V1 *-*- Q60D46_SOLDE)

129   136 4 Solyc10g006090.2.1 2583~2603(cDNA) Genomic DNA chromosome 5 P1  clone MTE17 (AHRD V1 **-- Q9FJ71_ARATH); contains Interpro domain(s) IPR011011 Zinc finger, FYVE/ PHD-type

129   137 3.5 Solyc12g044780.1.1 816~836(cDNA) F-box family protein (AHRD V1 *-*- D7LXD8_ARALY); contains Interpro domain(s) IPR001810 Cyclin-like F-box

129   138 3.5 Solyc12g044790.1.1 816~836(cDNA) F-box family protein (AHRD V1 *-*- D7LXD8_ARALY); contains Interpro domain(s) IPR001810 Cyclin-like F-box siR20 SIR2 LTR transposon TAGTGTTCTTGTTT TTCTGATT (SEQ ID 1402.4 467.4 83.2

139   140 4 AT3G18010.1 1076~1098(CDS) WUSCHEL related homeobox 1 NO: 139)

139   141 4.5 AT3G20660.1 43~65(5′UTR) organic cation/ carnitine  transporter4

139   142 4.5 AT4G23882.1 549~571(CDS) Heavy metal transport/ detoxification superfamily protein

139   143 4.5 AT5G17680.1 3220~3242(CDS) disease resistance protein (TIR-NBS-LRR class), putative

139   144 4.5 Solyc02g076690.2.1 598~620(cDNA) Cathepsin B-like  cysteine proteinase (AHRD V1 **-* CYSP_SCHMA);  contains Interpro domain(s) IPR013128 Peptidase C1A, papain

139   145 4.5 Solyc03g117110.2.1 462~484(cDNA) DCN1-like protein 4 (AHRD V1 ***- B6TI85_MAIZE);  contains Interpro domain(s) IPR014764  Defective in cullin neddylation

139   146 4.5 Solyc03g120530.2.1 163~185(cDNA) BHLH transcription factor-like protein (AHRD V1 *-** Q5ZAK6_ORYSJ); contains Interpro domain(s) IPR011598 Helix-loop-helix DNA-binding

139   147 4.5 Solyc11g039880.1.1 1821~1843(cDNA) Nucleoporin NUP188 homolog (AHRD V1  *-*- NU188_HUMAN) siR1021 SIR1021 CDS TACCAGTGATGAAC AAAACATGT (SEQ ID NO: 148) 2041.3 137.44 94.1

148   149 3 AT2G40520.1 815~837(CDS) Nucleotidyltransferase family protein

148   150 3.5 AT3G11530.1 682~704(3′UTR) Vacuolar protein sorting 55 (VPS55) family protein

148   151 4.5 Solyc05g009280.2.1 1339~1361(cDNA) Fatty acid elongase  3-ketoacyl-CoA synthase(AHRD V1 **** Q6DUV5_BRANA); contains Interpro domain(s) IPR012392 Very-long-chain 3- ketoacyl-CoA synthase siR1002 SIR1002 Intergenic region ATTCTTCAAATCTT TGTAACACA (SEQ ID 1408.4 360.44 239.1

152   153 4.5 AT1G62940.1 111~134(CDS) acyl-CoA synthetase 5 NO: 152)

152   154 4.5 AT4G30420.1 1039~1062(CDS) nodulin MtN21/EamA- like transporter family protein

152   155 3.5 AT4G34380.1 285~308(5′UTR) Transducin/WD40 repeat-like  superfamily protein

152   156 4 Solyc08g060920.2.1 98~121(cDNA) Xenotropic and  polytropic retrovirus receptor (AHRD V1 **-- B2GU54_XENTR); contains Interpro domain(s) IPR004331 SPX, N-terminal

152   157 4 Solyc08g081380.2.1 989~1012(cDNA) At5g63850- like protein (Fragment) (AHRD V1 *-*- Q3YI76_ARALY); contains Interpro domain(s) IPR000210 BTB/POZ-like

152   158 4.5 Solyc12g009480.1.1 67~90(cDNA) Xenotropic and  polytropic retrovirus receptor (AHRD V1 **-- B2GU54_XENTR); contains Interpro domain(s) IPR004331 SPX, N-terminal siR28 SIR1 LTR transposon TTTTTGAAACTGTG ATCTTCTT (SEQ ID 415.5 727.44 29.8

159   160 2.5 AT1G16760.1 1454~1476(CDS) Protein kinase protein with adenine nucleotide alpha hydrolases- NO: 159) like domain

159   161 2 AT1G78940.1 1425~1447(CDS) Protein kinase protein with adenine nucleotide alpha hydrolases- like domain

159   162 4 AT2G28830.1 2571~2593(CDS) PLANT U-BOX 12

159   163 4.5 AT2G40720.1 2191~2213(CDS) Tetratricopeptide  repeat (TPR)-like superfamily protein

159   164 4.5 AT3G20200.1 1777~1799(CDS) Protein kinase protein with adenine nucleotide alpha hydrolases- like domain

159   165 3 AT4G31230.1 1505~1527(CDS) Protein kinase protein with adenine nucleotide alpha hydrolases- like domain

159   166 4.5 Solyc01g080610.2.1 852~874(cDNA) Unknown Protein (AHRD V1); contains Interpro domain(s) IPR005508 Protein of unknown function DUF313

159   167 4.5 Solyc01g080720.2.1 319~341(cDNA) Pentatricopeptide  repeat-containing protein (AHRD V1 ***- D7L610_ARALY); contains Interpro domain(s) IPR002885 Pentatricopeptide repeat

159   168 4.5 Solyc03g115850.2.1 934~956(cDNA) NAC domain protein IPR003441 (AHRD V1 ***- B9I557_POPTR); contains Interpro domain(s) IPR003441 No apical meristem (NAM) protein

159   169 3 Solyc05g024450.1.1 196~218(cDNA) Unknown Protein (AHRD V1)

159   170 3.75 Solyc06g009200.2.1 664~686(cDNA) Polygalacturonase  (AHRD V1 ***- Q2M4X6_LILLO); contains Interpro domain(s) IPR000743 Glycoside hydrolase, family 28

159   171 4 Solyc06g031690.2.1 345~367(cDNA) Ankyrin repeat family protein (AHRD V1 ***- D7LCV0_ARALY); contains Interpro domain(s) IPR002110 Ankyrin

159   172 4 Solyc07g041780.2.1 450~472(cDNA) OBP3-responsive gene 4 (AHRD V1 **-- D7L9C5_ARALY) siR31 SIR1 LTR transposon TGAGTCTTGTGGTC GTGAATG (SEQ ID 117 803.16 4.7

173   174 4.5 AT1G65550.1 761~782(CDS) Xanthine/uracil permease family protein NO: 173)

173   175 4 AT2G05970.1 569~590(CDS) F-box family protein with a domain of unknown function (DUF295)

173   176 4.5 AT5G25420.1 716~737(CDS) Xanthine/uracil/ vitamin C permease

173   177 3.5 Solyc01g011090.2.1 3435~3456(cDNA) Phospholipid- transporting ATPase (AHRD V1 ***- C5G6U4_AJEDR): contains Interpro domain(s) IPR001757 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/ Ca/Na/H-transporter

173   178 4.5 Solyc01g110700.2.1 36445~36466(cDNA) Unknown Protein (AHRD V1)

173   179 4.5 Solyc01g111180.2.1 6734~6755(cDNA) Unknown Protein (AHRD V1) siR29 SIR2 LTR transposon TGTTGGATAGTCCT TTTTGGG (SEQ ID 1843 87.24 28.9

180   181 4.5 AT2G45110.1 729~750(CDS) expansin B4 NO: 180)

180   182 3.75 AT5G38990.1 1156~1177(CDS) Malectin/receptor- like protein kinase family protein

180   183 4 Solyc00g025660.1.1 576~597(cDNA) Unknown Protein (AHRD V1)

180   184 4 Solyc03g117510.2.1 745~766(cDNA) Formamidopyrimidine- DNA glycosylase (AHRD V1 **** C5JTH8_AJEDS); contains Interpro domain(s) IPR000191 DNA glycosylase/A P lyase siR41 SIR3 LTR transposon TGATAGTTTTCGGG AGTAGAA (SEQ ID 371.5 652.56 54.5

185   186 4.5 AT3G09530.1 826~847(CDS) exocyst subunit exo70 family protein H3 NO: 185)

185   187 4.5 AT3G19780.1 1248~1269(CDS)

185   188 4 Solyc05g014050.2.1 1422~1443(cDNA) Inner membrane protein oxaA (AHRD V1 *-*-  B9L0L4_THERP); contains Interpro domain(s) IPR001708 Membrane insertion protein, OxaA/YidC siR35 SIR2 LTR transposon TGTACTGTGCCATG TCGCGTT (SEQ ID 149.7 727.44 21.2

189   190 4 AT3G52810.1 978~999(CDS) purple acid phosphatase 21 NO: 189)

189   191 3.5 Solyc11g017230.1.1 721~742(cDNA) DNA polymerase I (AHRD V1 ***- B6U7X8_MAIZE); contains Interpro domain(s) IPR002421 5'-3' exonuclease, N-terminal siR57 SIR1 LTR transposon TAGATAATCTCTG GTTCGTTGG (SEQ ID 114 728.28 13.8

192   193 4.5 AT3G28390.1 3253~3275(CDS) P-glycoprotein 18 NO: 192)

192   194 4.5 AT3G29575.1 350~372(CDS) ABI five binding protein 3

192   195 2.5 Solyc03g007790.2.1 2084~2106(cDNA) Receptor-like protein kinase (AHRD V1 ****  Q9FLV4_ARATH); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase siR43 SIR1 LTR transposon TGGGAGCTTTCTC TTGTTGGG (SEQ ID 645 501.16 122

196   197 4 AT1G19050.1 592~613(CDS) response regulator 7 NO: 196)

196   198 4.5 AT1G26450.1 401~422(CDS) Carbohydrate-binding  X8 domain superfamily protein

196   199 4.5 AT1G51600.1 1398~1419(3'UTR) ZIM-LIKE 2

196   200 4.25 AT1G70190.1 202~223(CDS) Ribosomal protein L7/L12,  oligomerisation; Ribosomal protein L7/L12, C-terminal/ adaptor protein ClpS-like

196   201 4.25 AT3G19860.1 979~1000(CDS) basic helix-loop- helix (bHLH) DNA- binding superfamily protein

196   202 4.5 AT5G45030.1 65~86(5'UTR) Trypsin family protein

196   203 4.5 Solyc01g093970.2.1 809~830(cDNA) Glycosyltransferase (AHRD V1 **-- B9IC41_POPTR); contains Interpro domain(s) IPR002495 Glycosyl transferase, family 8

196   204 3.5 Solyc04g039950.2.1 2037~2058(cDNA) Mediator of RNA polymerase II transcription subunit 13 (AHRD V1 *-*- MED13_DICDI); contains Interpro domain(s) IPR009401 Mediator complex, subunit Med13 siR40 SIR2 LTR transposon TGGAATGGGCTTG TATTGGTT (SEQ ID 693.6 473.16 43

205   206 4.5 AT1G06910.1 756~777(CDS) TRF-like 7 NO: 205)

205   207 4.5 AT1G09350.1 723~744(CDS) galactinol synthase 3

205   208 3.5 AT4G38550.1 604~625(CDS) Arabidopsis phospholipase-like protein (PEARLI 4) family

205   209 4 Solyc02g037560.1.1 542~563(cDNA) Ulp1 protease family  C-terminal catalytic domain containing protein (AHRD V1 ***- Q60D46_SOLDE)

205   210 4 Solyc08g074820.1.1 86~107(cDNA) Unknown Protein (AHRD V1) siR38 SIR2 LTR transposon TAATTCAGGAGAC GATATCGT (SEQ ID 1765.5 35.4 23.3

211   212 4.5 AT3G23130.1 1039~1060(3'UTR) C2H2 and C2HC zinc fingers superfamily protein NO: 211)

211   213 4.25 Solyc04g081500.2.1 836~857(cDNA) BRCA1-A complex subunit BRE (AHRD V1 ***- BRE_XENTR); contains Interpro domain(s) IPR010358 Brain and reproductive organ- expressed siR46 SIR9  Intergenic region CTAACGATTGAA GGCCACCAAC (SEQ ID 1811.1 5.76 166.5

214   215 4 AT5G21430.1 703~725(CDS) Chaperone DnaJ- domain superfamily protein NO: 214)

214   216 3 Solyc09g007340.2.1 938~960(cDNA) PWWP domain- containing protein (AHRD V1 *-*- D7L8B3_ARALY); contains Interpro domain(s) IPR000313 PWWP siR48 SIR1 LTR transposon TGAAGTGACAGT ATCGATCAA (SEQ ID 66.9 678.08 7.7

217   218 4 AT2G03040.1 444~465(CDS) emp24/gp25L/p24 family/GOLD family protein NO: 217)

217   219 4 AT2G03290.1 444~465(CDS) emp24/gp25L/p24 family/GOLD family protein

217   220 4 AT2G44430.1 511~532(CDS) DNA-binding  bromodomain- containing protein

217   221 4.5 AT5G58160.1 1894~1915(CDS) actin binding

217   222 3 Solyc06g068240.2.1 441~462(cDNA) Pyrophosphate- energized proton pump (AHRD V1 ***- B0SRX3_LEPBP);  contains Interpro domain(s) IPR004131 Inorganic H+ pyrophosphatase

217   223 4.5 Solyc12g099250.1.1 1641~1662(cDNA) Kinase family protein (AHRD V1 ***- D7KVQ9_ARALY); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase siR1007 SIR1007 LTR transposon GTAGGTGATCCTG CGGAAGGAT (SEQ ID 1641.7 14 0

224   225 4.5 AT3G09370.1 334~356(CDS) myb domain protein 3r-3 NO: 224)

224   226 3 Solyc12g099450.1.1 514~536(cDNA) Genomic DNA chromosome 5 TAC clone K20J1(AHRD  V1 *-*- Q9FH24_ARATH) siR56 SIR1 LTR transposon TCGTTCATCCTGTA GTTGCGT (SEQ ID 38.7 655.04 19.1

227   228 4 AT5G37010.1 1380~1401(CDS) unknown protein, hypothetical protein, uncharacterized protein NO: 227)

227   229 4 Solyc03g019870.2.1 915~936(cDNA) Cytochrome P450 siR49 SIR2 LTR transposon TGTGGCTTATGTCT TTTGATA (SEQ ID 1079.5 228.76 50.6

230   231 4.5 AT3G45700.1 1535~1556(CDS) Major facilitator superfamily protein NO: 230)

230   232 4.5 AT4G01410.1 940~961(3′UTR) Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

230   233 4.5 Solyc01g107100.2.1 82~103(cDNA) Beta-1,4-xylosidase (AHRD V1 ***- D7LA14_ARALY)

230   234 4.25 Solyc07g042160.2.1 1440~1461(cDNA) Polygalacturonase  (AHRD V1 **-- B6SZN5_MAIZE); contains Interpro domain(s) IPR012334 Pectin lyase fold siR58 SIR1 LTR transposon TAAATTGGGATTCA TTGTCTG (SEQ ID 39.5 636.12 7

235   236 4.5 AT4G36080.1 4572~4593(CDS) phosphotransferases, alcohol group as acceptor; binding; inositol or NO: 235) phosphatidylinositol kinases

235   237 4.5 Solyc01g058540.2.1 1023~1044(cDNA) WRKY transcription factor 31 (AHRD  V1 *-*-  C9DI20_9ROSI); contains Interpro domain(s) IPR003657 DNA-binding WRKY

235   238 4 Solyc01g109980.2.1 2186~2207(cDNA) BEL1-like homeodomain protein 6 (AHRD V1 *--- BLH6_ARATH); contains Interpro domain(s) IPR006563 POX siR63 SIR1 LTR transposon TAATAGTTGATGA GAGAATGT (SEQ ID 132.9 578.48 7.8

239   240 4.5 AT5G04430.1 1461~1482(3′UTR) binding to TOMV RNA 1L (long form) NO: 239)

239   241 4.5 AT5G48385.1 2124~2145(3′UTR) FRIGIDA-like protein

239   242 4.5 Solyc01g096910.2.1 975~996(cDNA) Vacuolar protein sorting 36 family protein (AHRD V1 ***- D7LY74_ARALY); contains Interpro domain(s) IPR007286 EAP30 siR1005 SIR1005 LTR transposon TAAAGAGTTTCTT CAATAGGA (SEQ ID 441.4 452.6 277.5

243   244 4 AT1G20200.1 1224~1245(CDS) PAM domain (PCI/PINT associated module) protein NO: 243)

243   245 4.5 AT1G20650.1 1502~1523(CDS) Protein kinase superfamily protein

243   246 4.5 AT1G67540.1 352~373(CDS) unknown protein, hypothetical protein, uncharacterized protein

243   247 4.5 AT2G23790.1 82~103(CDS) Protein of unknown function (DUF607)

243   248 4.5 AT3G50950.1 2116~2137(CDS) HOPZ-ACTIVATED RESISTANCE 1

243   249 4.5 AT4G14510.1 1862~1883(CDS) CRM family member 3B

243   250 4.5 AT5G61290.1 1366~1387(CDS) Flavin-binding monooxygenase family protein

243   251 3.5 Solyc01g091200.2.1 824~845(cDNA) NAD dependent epimerase/dehydratase family protein expressed (AHRD V1 ***- Q2MJA7_ORYSJ); contains Interpro domain(s) IPR016040 NAD(P)-binding domain

243   252 4.5 Solyc04g028560.2.1 2604~2625(cDNA) Zinc finger transcription factor  (AHRD V1 *-** Q7K9G4_DROME); contains Interpro domain(s) IPR013087 Zinc finger, C2H2-type/integrase, DNA-binding

243   253 3.5 Solyc05g050990.1.1 478~499(cDNA) UDP-D-glucuronate  4-epimerase 2 (AHRD V1 **** D7M5S7_ARALY); contains Interpro domain(s) IPR016040 NAD(P)-binding domain

243   254 4.5 Solyc10g005940.1.1 191~212(cDNA) CT099 (Fragment) (AHRD V1 *--- Q4KR02_SOLCI); contains Interpro domain(s) IPR003245 Plastocyanin-like siR60 SIR1 LTR transposon TGCAATGGAATTCG AAGACGG (SEQ ID 33.4 599.88 34.1

255   256 4.5 AT1G55610.1 817~838(CDS) BRI1 like NO: 255)

255   257 4.25 Solyc08g067800.1.1 261~282(cDNA) Acetyltransferase (AHRD V1 **-* B4RG69_PHEZH); contains Interpro domain(s) IPR016181 Acyl-CoA N-acyltransferase siR61 SIR3 LTR transposon TAGAATAGAATC GTATACGTG (SEQ ID 230.9 515.12 10.3

258   259 4.25 AT2G17510.2 1543~1564(CDS) ribonuclease II family protein NO: 258)

258   260 4 Solyc03g078160.2.1 896~917(cDNA) POT family domain containing protein expressed (AHRD V1 ***- D8L9H8_WHEAT); contains Interpro domain(s) IPR007493 Protein of unknown function DUF538

258   261 3.5 Solyc03g121810.2.1 2888~2909(cDNA) Phospholipid- transporting ATPase 1 (AHRD V1 **** C5FPS3_NANOT); contains Interpro domain(s) IPR006539 ATPase, P-type, phospholipid- translocating, flippase

258   262 4.5 Solyc04g082430.2.1 8~29(cDNA) B-like cyclin (AHRD V1**** Q40337_MEDSA); contains Interpro domain(s) IPR014400 Cyclin, A/B/D/E siR62 SIR2 LTR transposon TACGACGGATTCG CAAGTAAA (SEQ ID 149.7 547.24 8.6

263   264 4.25 AT1G11620.1 353~374(CDS) F-box and associated interaction domains- containing protein NO: 263)

263   265 4 AT4G10030.1 100~121(5′UTR) alpha/beta- Hydrolases  superfamily protein

263   266 4 Solyc01g009570.2.1 236~257(cDNA) Unknown Protein (AHRD V1) siR65 SIR1 LTR transposon TAGCAAGAGGGA TTCTGTAGT (SEQ ID 14.4 583.44 22.2

267   268 4 AT1G75950.1 282~303(CDS) S phase kinase- associated protein 1 NO: 267)

267   269 4 AT2G21330.1 974~995(CDS) fructose-bisphosphate aldolase 1

267   270 4.5 AT3G23670.1 3292~3313(CDS) phragmoplast- associated kinesin-related protein, putative

267   271 4.5 AT4G25980.1 187~208(CDS) Peroxidase superfamily protein

267   272 4.5 AT4G27680.1 1507~1528(3′UTR) P-loop containing nucleoside  triphosphate hydrolases  superfamily protein

267   273 4 Solyc07g007790.2.1 3439~3460(cDNA) Sucrose phosphate synthase (AHRD V1 **** Q2HYI0_CUCME); contains Interpro domain(s) IPR012819 Sucrose phosphate synthase, plant

267   274 4.5 Solyc12g008370.1.1 496~517(cDNA) Pre-mRNA-processing protein 45 (AHRD V1 **-- D6RKF6_COPC7); contains Interpro domain(s) IPR017862 SKI-interacting protein, SKIP siR67 SIR2 LTR transposon TAAATCGATCGGA GAATTTTTT (SEQ ID 687.5 297.88 25.7

275   276 4 AT1G27880.1 3~25(CDS) DEAD/DEAH (SEQ ID NOS: 277 and 278)  box RNA helicase family protein NO: 275)

275   279 3 Solyc05g055050.1.1 568~590(cDNA) Calcium-dependent protein kinase 2  (AHRD V1 **** B4FZS4_MAIZE); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase

275   280 4 Solyc07g053900.2.1 421~443(cDNA) Plant-specific domain TIGR01615 family protein (AHRD V1 *-*- B6UDN7_MAIZE); contains Interpro domain(s) IPR006502 Protein of unknown function DUF506, plant siR68 SIR1 LTR transposon TGGATGCAGTGATC GGAATTG (SEQ ID 20.5 534.88 6.4

281   282 4.25 AT4G21700.1 167~188(CDS) Protein of unknown function (DUF2921) NO: 281)

281   283 4 Solyc04g009560.2.1 2811~2832(cDNA) TBC1 domain family member 8B (AHRD V1  *--- B9A6K5_HUMAN); contains Interpro domain(s) IPR000195 RabGAP/TBC

281   284 4.5 Solyc10g007340.2.1 453~474(cDNA) Unknown Protein (AHRD V1) siR73 SIR3 LTR transposon TGTGCCCAATCTAT TTTCGGA (SEQ ID 478.6 305.28 141.6

285   286 4 AT1G17020.1 459~480(CDS) senescence-related gene 1 NO: 285)

285   287 4.5 Solyc01g111250.2.1 533~554(cDNA) Phosphatidylinositol- specific phospholipase c (AHRD V1 *-*- B9UXN2_LISMO); contains Interpro domain(s) IPR017946 PLC-like phosphodiesterase, TIM beta/alpha- barrel domain

285   288 4.5 Solyc01g111260.2.1 543~564(cDNA) Phosphatidylinositol- specific phospholipase c (AHRD V1 *-*- B9UY71_LISMO); contains Interpro domain(s) IPR017946 PLC-like phosphodiesterase, TIM beta/alpha- barrel domain

285   289 4.5 Solyc06g069280.2.1 1359~1380(cDNA) Protein LSM14 homolog A (AHRD V1  *--- LS14A_PONAB); contains Interpro domain(s) IPR019053 FFD and TFG box motifs siR81 SIR1 LTR transposon TGTCTCTAATCAA GCGTTGGG (SEQ ID 28.1 438.6 3.6

290   291 4.5 AT5G48670.1 403~424(CDS) AGAMOUS-like 80 NO: 290)

290   292 4.5 Solyc03g082940.2.1 1376~1397(cDNA) Importin subunit beta (AHRD V1 ***- B0WBR4_CULQU); contains Interpro domain(s) IPR011989 Armadillo-like helical

290   293 4.5 Solyc08g062940.2.1 810~831(cDNA) Calmodulin binding protein (AHRD V1 **-* B6T951_MAIZE); contains Interpro domain(s) IPR000048 IQ calmodulin- binding region siR82 SIR1 LTR transposon TGATACGGATTTCT TAACTGAT (SEQ ID 275 335.76 26.9

294   295 4.5 AT2G45540.1 4598~4620(CDS) WD-40 repeat family protein/beige-related NO: 294)

294   296 4 Solyc11g006560.1.1 922~944(cDNA) Glycosyl transferase group 1 (AHRD V1 ***- B6T775_MAIZE); contains Interpro domain(s) IPR001296 Glycosyl transferase, group 1 siR86 SIR2 LTR transposon TGTTGATAGCTGAT TTGATGGT (SEQ ID 695.9 147.28 89.9

297   298 3.25 AT1G10180.1 2187~2209(CDS) uncharacterized protein. hypothetical protein NO: 297)

297   299 4.5 AT5G66650.1 734~756(CDS) Protein of unknown function (DUF607)

297   300 4.5 Solyc01g058190.2.1 1101~1123(cDNA) 30S ribosomal protein  S6 (AHRD V1 *-*- B4WXW0_9GAMM); contains Interpro domain(s) IPR000529 Ribosomal protein S6

297   307 4.5 Solyc05g052280.2.1 211~233(cDNA) Peroxidase (AHRD V1 ***- B9VRK9_CAPAN); contains Interpro domain(s) IPR002016 Haemperoxidase, plant, fungal/ bacterial siR91 SIR2 LTR transposon TGGTGCTGTTGATA GCTGATTT (SEQ ID 533.3 187.64 32.5

302   303 4 AT1G70620.1 654~676(CDS) cyclin-related NO: 302)

302   304 4.5 Solyc01g006030.2.1 449~471(cDNA) E3 ubiquitin-protein ligase bre1 (AHRD V1  *-*- B6K254_SCHJY); contains Interpro domain(s) IPR018957 Zinc finger, C3HC4 RING-type

302   305 4.5 Solyc01g060270.1.1 975~997(cDNA) Os06g0207500 protein (Fragment)(AHRD V1 ***- Q0DDQ9_ORYSJ); contains Interpro_ domain(s) IPR004253 Protein of unknown function DUF231, plant

302   306 4 Solyc05g026330.1.1 322~344(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   307 4 Solyc05g026350.1.1 444~466(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   308 4 Solyc05g041300.1.1 183~205(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   309 4.5 Solyc05g041320.1.1 322~344(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   310 4.5 Solyc05g041610.1.1 415~437(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   311 4.5 Solyc05g041620.1.1 322~344(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3

302   312 4 Solyc05g041690.1.1 475~497(cDNA) Caffeoyl-CoA O-methyltransferase (AHRD V1 **** A2PZD5_IPONI); contains Interpro domain(s) IPR002935 O-methyltransferase, family 3 siR92 SIR3 LTR transposon TGTACTGTTCTGGT ATCGTAGG (SEQ ID 29.6 374.44 22.5

313   314 3.5 AT3G45620.1 701~723(CDS) Transducin/WD40  repeat-like superfamily protein NO: 313)

313   315 4 Solyc02g085760.2.1 491~513(cDNA) Rhomboid family protein (AHRD V1 ***- D7MJX8_ARALY); contains Interpro domain(s) IPR002610 Peptidase S54, rhomboid siR95 SIR1 LTR transposon TGCGAAGTTATGT ATAGTAGA (SEQ ID 20.5 373.6 3.2

316   317 4.5 AT2G03060.1 1405~1426(3′UTR) AGAMOUS-like 30 NO: 316)

316   318 4 Solyc08g016050.2.1 1697~1718(cDNA) Dedicator of cytokinesis family protein (AHRD V1 ***- A8P5S7_BRUMA);  contains Interpro domain(s) IPR010703 Dedicator of cytokinesis siR1017 SIR1017 Intergenic region AGGGTGGAGAGA GTTCGGACATTC 711.8 95.44 113.1

319   320 4.5 AT3G11910.1 1418~1442(CDS) ubiquitin-specific protease 13 (SEQ ID NO: 319)

319   321 4.5 Solyc03g007760.2.1 1996~2020(cDNA) Cell division protease  ftsH (AHRD V1  *--- FTSH_SHIFL); contains Interpro domain(s) IPR003959 ATPase, AAA-type, core siR97 SIR3 LTR transposon TATCGGGTCCATCC TTCTTGGG (SEQ ID 114 331.64 40.2

322   323 4.5 AT4G17505.1 185~207(CDS) Protein of Unknown Function (DUF239) NO: 322)

322   324 4.5 Solyc01g091370.2.1 1179~1201(cDNA) AT-hook motif nuclear localized protein 1 (AHRD V1 ***- Q8VYJ2_ARATH); contains Interpro domain(s) IPR005175 Protein of unknown function DUF296

322   325 3 Solyc01g094640.2.1 2690~2712(cDNA) uncharacterized protein LOC101249582 (related) (AHRD V1 ***- Q2HTJ8_MEDTR) siR99 SIR2 LTR transposon TAGTGTCAGCTAAT TCAGGAG (SEQ ID 366.9 216.44 13.1

326   327 4.5 AT2G07360.1 412~433(CDS) SH3 domain-containing proteins NO: 326)

326   328 4.5 AT2G39100.1 1127~1148(3′UTR) RING/U-box superfamily protein

326   329 3 AT5G13320.1 889~910(CDS) Auxin-responsive GH3 family protein

326   330 4.5 Solyc02g067320.1.1 52~73(cDNA) Zinc finger- homeodomain protein 1 (Fragment) (AHRD V1 **-- B0LK19_CUCSA); contains Interpro domain(s) IPR006456 ZF-HD homeobox protein Cys/His-rich dimerisation region

326   331 4.5 Solyc08g066940.2.1 1557~1578(cDNA) Peptide transporter 1 (AHRD V1 **-* Q7XAC3_VICFA); contains Interpro domain(s) IPR000109 TGF-beta receptor, type I/II extracellular region siR1013 SIR1013 CDS TTATATGATGAAC AAACTTTAAA (SEQ 521.1 149.76 24.4

332   333 4.5 AT1G79840.1 77~100(5′UTR) HD-ZIP IV family of homeobox-leucine zipper protein with lipid- binding START domain ID NO: 332)

332   334 4 Solyc03g098070.2.1 1258~1281(cDNA) C2H2L domain class transcription factor (AHRD V1 *--* D9ZIU3_MALDO); contains Interpro domain(s) IPR007087 Zinc finger, C2H2-type siR102 SIR13 Intergenic region TGGAGGGGAGAT TGATACATTG (SEQ 827.3 20.56 101.5

335   336 3.5 AT3G13750.1 3258~3280(3′UTR) beta galactosidase 1 ID NO: 335)

335   337 4.5 AT5G43100.1 139~161(CDS) Eukaryotic aspartyl protease family protein

335   338 4 Solyc11g067000.1.1 2884~2906(cDNA) ATP-binding cassette transporter (AHRD V1 ***- D8T797_SELML); contains Interpro domain(s) IPR013525 ABC-2 type transporter siR1011 SIR1011 CDS  TAATATGATGAGC AAGATTGGT (SEQ ID 413.3 172.8 117.2

339   340 4.5 AT4G21215.1 724~746(CDS) NO: 339)

339   341 3 AT5G51530.1 3078~3100(CDS) Ubiquitin carboxyl- terminal hydrolase- related protein

339   342 4 AT5G67140.1 772~794(CDS) F-box/RNI-like superfamily protein

339   343 4.5 Solyc02g093150.2.1 1404~1426(cDNA) AP2-like ethylene- responsive  transcription factor At1g16060 (AHRD V1 *-*- AP2L1_ARATH); contains Interpro domain(s) IPR001471 Pathogenesis-related transcriptional factor and ERF, DNA- binding siR109 SIR3 LTR transposon TGCTGGTGTGATTT TCGTGGT (SEQ ID 437.6 160.48 150

344   345 4.5 AT5G64390.1 377~398(CDS) RNA-binding KH domain- containing protein NO: 344)

344   346 4.5 Solyc01g103550.2.1 2540~2561(cDNA) Cell division protein kinase 13 (AHRD V1 *-*- CDK13_MOUSE); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase

344   347 4.25 Solyc02g069630.2.1 2706~2727(cDNA) Subtilisin-like serine protease (AHRD V1 **-* Q948Q4_ARATH); contains Interpro domain(s) IPR015500 Peptidase S8, subtilisin-related

344   348 3.5 Solyc05g015510.2.1 3013~3034(cDNA) Squamosa promoter- binding-like protein 11 (AHRD V1 ***- B6TF72_MAIZE); contains Interpro domain(s) IPR004333 Transcription factor, SBP-box

344   349 4.5 Solyc09g007710.2.1 3351~3372(cDNA) Tir-nbs-lrr, resistance protein

344   350 4 Solyc10g081020.1.1 3688~3709(cDNA) Transcription elongation factor SPT6 (AHRD V1 ***- A8NF94_COPC7); contains Interpro domain(s) IPR017072 Transcription elongation factor Spt6 siR1018 SIR8 Intergenic region TGATGTTGCATACC CGGCTCGG (SEQ ID 618.4 51 288.2

351   352 4.5 AT1G62970.1 1017~1039(CDS) Chaperone DnaJ-domain superfamily protein NO: 351)

351   353 4.5 Solyc04g007510.2.1 3230~3252(cDNA) ATP-dependent RNA helicase A-like  protein (AHRD V1 ***- Q9FF84_ARATH); contains Interpro domain(s) IPR007502 Helicase- associated region siR114 SIR2 LTR transposon TCCAGGGTCCTTT TGGAATAGG (SEQ ID 395.8 138.24 14.3

354   355 4.5 AT1G78960.1 1445~1467(CDS) lupeol synthase 2 NO: 354)

354   356 4.5 Solyc12g006510.1.1 1377~1399(cDNA) Cycloartenol Synthase (AHRD V1 ***- O82139_PANGI);  contains Interpro domain(s) IPR018333 Squalene cyclase siR1020 SIR1020 Intergenic region TTGCCACGACGAA CCAGGACA (SEQ ID 138.3 209 10.1

357   358 4 AT2G22810.1 1176~1197(CDS) 1-aminocyclopropane- 1-carboxylate synthase 4 NO: 357)

357   359 4 Solyc04g005650.1.1 337~358(cDNA) Mitochondrial carrier family (AHRD V1 ***- C1MWU5_MICPS); contains Interpro domain(s) IPR001993 Mitochondrial substrate carrier

357   360 4.5 Solyc09g091210.2.1 861~882(cDNA) Disease resistance response/dirigent-like protein (AHRD V1 ***- Q0WPQ6_ARATH); contains Interpro domain(s) IPR004265 Plant disease resistance response protein siR1016 SIR1 LTR transposon TTGAGAGCTAAGT CAAACGGA (SEQ ID 22.8 255.08 5

361   362 3.5 AT1G23190.1 1753~1774(CDS) Phosphoglucomutase/ phosphomannomutase family protein NO: 361)

361   363 4.25 AT5G19260.1 184~205(CDS) Protein of unknown function (DUF3049)

361   364 3.5 Solyc01g101090.2.1 1040~1061(cDNA) TBC1 domain family member CG11727 (AHRD V1 **-* Y1727_DROME); contains Interpro domain(s) IPR000195 RabGAP/TBC

361   365 4 Solyc02g082060.1.1 497~518(cDNA) PPPDE peptidase domain-containing protein 1 (AHRD V1  *--- PPDE1_XENLA); contains Interpro domain(s) IPR008580 Protein of unknown function DUF862, eukaryotic

361   366 3.5 Solyc04g076690.2.1 623~644(cDNA) Unknown Protein (AHRD V1) siR1003 SIR1003 LTR transposon GGTAACCAGAAC TGGCGATGC (SEQ ID 615.3 2.48 0.5

367   368 4 AT2G31220.1 223~244(CDS) basic helix-loop-helix (bHLH) DNA-binding superfamily protein NO: 367)

367   369 4 Solyc06g050170.2.1 1771~1792(cDNA) Potassium transporter (AHRD V1 **** Q1T761_PHRAU); contains Interpro domain(s) IPR018519 Potassium uptake protein, kup IPR003855 K+ potassium transporter siR124 SIR1 LTR transposon TGACCAGAGCTCC GGGGAGGT (SEQ ID 17.5 232.88 3.6

370   371 4 AT1G13270.1 140~161(CDS) methionine aminopeptidase 1B NO: 370)

370   372 4.5 AT3G59040.1 1252~1273(CDS) Tetratricopeptide repeat (TPR)-like superfamily protein

370   373 4.5 Solyc02g065550.2.1 280~301(cDNA) Coiled-coil domain- containing protein  109A (AHRD V1 *--- C109A_MOUSE); contains Interpro domain(s) IPR006769 Protein of unknown function DUF607

370   374 4 Solyc04g045540.1.1 127~148(cDNA) Ycfl (Fragment) (AHRD V1 ***- A6YA36_9MAGN); contains Interpro domain(s) IPR008896 Ycfl

370   375 4 Solyc05g047440.1.1 127~148(cDNA) Ycfl (Fragment) (AHRD V1 ***- A6Y9X6_HAMJA); contains Interpro domain(s) IPR008896 Ycfl

370   376 4.25 Solyc05g055360.2.1 1577~1598(cDNA) Unknown Protein  (AHRD V1)

370   377 4 Solyc10g062330.1.1 82~103(cDNA) Hypothetical chloroplast RF1 (AHRD V1 **-- C3UP30_9MAGN); contains Interpro domain(s) IPR008896 Ycfl

370   378 4 Solyc11g021310.1.1 127~148(cDNA) Hypothetical chloroplast RF1 (AHRD V1 C3UP30_9MAGN); contains Interpro domain(s) IPR008896 Ycfl siR127 SIR2 LTR transposon TGTTTTGACATGTT GTTTGACG (SEQ ID 451.3 54.32 19.2

379   380 4 AT5G10450.3 932~954(3′UTR) G-box regulating factor 6 NO: 379)

379   381 4.5 Solyc01g068430.1.1 871~893(cDNA) Os06g0207500 protein (Fragment) (AHRD V1 **-- Q0DDQ9_ORYSJ); contains Interpro domain(s) IPR004253 Protein of unknown function DUF231, plant siR128 SIR15  Intergenic region TACAGAATACAG AATCAAGAT (SEQ ID 574.3 3.28 7.7

382   383 3 AT1G48210.1 1343~1364(3′UTR) Protein kinase superfamily protein NO: 382)

382   384 4 AT2G23348.1 402~423(3′UTR) unknown protein, hypothetical protein, uncharacterized protein

382   385 3 AT4G08990.1 2536~2557(CDS) DNA (cytosine-5-)- methyltransferase family protein

382   386 4 AT4G14140.1 2560~2581(CDS) DNA methyltransferase 2

382   387 4.5 Solyc04g005530.2.1 1196~1217(cDNA) Unknown Protein (AHRD V1)

382   388 4.5 Solyc11g012550.1.1 49~70(cDNA) F-box family protein (AHRD V1 ***- D7L4T6_ARALY); contains Interpro domain(s) IPR001810 Cyclin-like F-box siR130 SIR2 LTR transposon TGTTCAACAAGTCT ATATTGGT (SEQ ID 400.4 65 6.5

389   390 4 AT2G42340.1 486~508(CDS) unknown protein, hypothetical protein, uncharacterized  protein NO: 389)

389   391 4 Solyc01g008080.2.1 2214~2236(cDNA) Ribosomal protein S27 (AHRD V1 ***- Q3HVK9_SOLTU); contains Interpro domain(s) IPR000592 Ribosomal protein S27e

389   392 3.5 Solyc01g095740.2.1 2485~2507(cDNA) ATP-dependent RNA helicase DBP4 (AHRD V1 *-**  C1GZM0_PARBA); contains Interpro domain(s) IPR011545 DNA/RNA helicase, DEAD/DEAH box type, N-terminal siR1004 SIR15 Intergenic region AATGATTGGAAGG AAGGAGTTC (SEQ ID 485.4 14 32.8

393   394 4.5 AT3G07990.1 72~94(CDS) serine  carboxypeptidase-like 27 NO: 393)

393   395 4.5 AT4G21740.1 99~121(CDS) unknown protein, hypothetical protein, uncharacterized  protein

393   396 4.25 Solyc07g042910.2.1 1930~1952(cDNA) Genomic DNA chromosome 5 TAC clone K21L19 (AHRD V1 **-- Q9FGT4_ARATH) siR144 SIR6 CDS (spurious gene) TAACATGATGATTA ATTTATC (SEQ ID 471 9.88 46.1

397   398 4 AT2G46330.1 471~492(3′UTR) arabinogalactan protein 16 NO: 397)

397   399 4 AT4G12040.2 513~534(5′UTR) A20/AN1-like zinc finger family protein

397   400 4.25 Solyc01g080260.2.1 2174~2195(cDNA) At4g14280-like protein (Fragment) (AHRD V1  *-*- C7FD87_ARALP); contains Interpro domain(s) IPR011989 Armadillo-like helical

397   401 4.5 Solyc01g098240.1.1 3823~3844(cDNA) RNA polymerase Rpb1 C-terminal repeat domain- containing protein (AHRD V1 *--- C5GU31_AJEDR); contains Interpro domain(s) IPR012474 Frigida-like

397   402 4.5 Solyc10g005650.2.1 814~835(cDNA) Peroxisomal targeting signal 1 receptor (AHRD V1 **** Q9ZTK6_TOBAC); contains Interpro domain(s) IPR011990 Tetratricopeptide- like helical

397   403 4.5 Solyc12g007150.1.1 73~94(cDNA) Pollen-specific kinase partner protein-like protein (Fragment) (AHRD V1 *--- Q5DK68_SOLLC); contains Interpro domain(s) IPR005512 Rop nucleotide exchanger, PRONE siR137 SIR2 LTR transposon TACGATTCTATTCT AGTAGTA (SEQ ID 376.8 46.08 3

404   405 4 AT1G22110.1 1283~1304(3′UTR) structural constituent of ribosome NO: 404)

404   406 4.5 AT3G25510.1 5473~5494(CDS) disease resistance protein (TIR-NBS-LRR class), putative

404   407 4.5 Solyc04g063230.2.1 1354~1375(cDNA) Dehydration-responsive family protein (AHRD V1 **-- D7LF23_ARALY); contains Interpro domain(s) IPR004159 Protein of unknown function DUF248, methyl transferase putative siR140 SIR8  Intergenic region TTGATTTTGCCGTT TCGTATGT (SEQ ID 417.1 27.16 49.1

408   409 4.5 AT2G07360.1 3291~3313(CDS) SH3 domain-containing protein NO: 408)

408   410 4.25 Solyc04g080720.2.1 1084~1106(cDNA) Transferase family protein (AHRD V1 **-* D7KBT0_ARALY); contains Interpro domain(s) IPR003480 Transferase

408   411 4 Solyc07g017860.2.1 436~458(cDNA) Acetyl-coenzyme A synthetase (AHRD V1 ***- Q2J3D0_RHOP2); contains Interpro domain(s) IPR011904 Acetate--CoA ligase

408   412 3.5 Solyc12g098610.1.1 641~663(cDNA) Xyloglucan endotransglucosylase/ hydrolase 8 (AHRD V1 ***- C0IRG7_ACTDE); contains Interpro domain(s) IPR016455 Xyloglucan endotransglucosylase/ hydrolase siR141 SIR1 LTR transposon TAGAAACATTCGG ACTTCTGT (SEQ ID 11.4 187.64 3

413   414 4 AT3G01350.1 1191~1212(CDS) Major facilitator superfamily protein NO: 413)

413   415 4.5 Solyc03g113070.2.1 1358~1379(cDNA) ATP-binding cassette (ABC) transporter 17 (AHRD V1 *-*- Q4H493_RAT) siR156 SIR18 Intergenic region TGGGATGGGATG GGATTGGGA (SEQ ID  335 9.88 251.1

416   417 4.5 AT5G45973.1 62~83(CDS) unknown protein, hypothetical protein, uncharacterized  protein NO: 416)

416   418 2 Solyc01g112220.2.1 163~184(cDNA) Serine/threonine protein kinase-like (AHRD V1 **** Q5XWQ1_SOLTU); contains Interpro domain(s) IPR002290 Serine/threonine protein kinase

416   419 4.5 Solyc12g019040.1.1 100~121(cDNA) Exostosin family  protein (AHRD V1  *-*- D7LPB7_ARALY)

416   420 4.5 Solyc12g096410.1.1 54~75(cDNA) Unknown Protein (AHRD V1) siR161 SIR1 LTR transposon TAGGCATCATTCTC TTCCTTGG (SEQ ID 9.9 120.16 5.7

421   422 4.5 AT2G16270.1 295~317(CDS) NO: 421)

421   423 4.5 AT3G18660.1 1168~1190(CDS) plant glycogenin- like starch initiation protein 1

421   424 4 AT3G63380.1 1416~1438(CDS) ATPase E1-E2 type family protein/ haloacid dehalogenase-like hydrolase family protein

421   425 3.75 AT5G17400.1 863~885(CDS) endoplasmic reticulum- adenine nucleotide transporter 1

421   426 2.5 Solyc03g083340.1.1 1152~1174(cDNA) Response regulator 8 (AHRD V1 *-*- Q9AV93_MAIZE); contains Interpro domain(s) IPR001789 Signal transduction response regulator, receiver region

421   427 4.5 Solyc04g005430.2.1 1312~1334(cDNA) Dehydration-responsive protein-like (AHRD V1 **-- Q653G1_ORYSJ); contains Interpro domain(s) IPR004159 Protein of unknown function DUF248, methyltransferase putative

421   428 4.5 Solyc11g005760.1.1 892~914(cDNA) Glycogenin-like protein (AHRD V1 ***- Q5NA53_ORYSJ); contains Interpro domain(s) IPR002495 Glycosyl transferase, family 8 siR163 SIR8 Intergenic region TGATCCAAAGTAC AATGTGTA (SEQ ID 275 8.24 74.2

429   430 2.5 AT3G07140.1 1754~1775(CDS) GPI transamidase component Gpi16 subunit family protein NO: 429)

429   431 4.5 AT5G46640.1 1159~1180(CDS) AT hook motif DNA-binding family protein

429   432 4.5 AT5G59810.1 293~314(CDS) Subtilase family protein

429   432 4.5 Solyc06g084310.2.1 598~619(cDNA) Small nuclear ribonucleoprotein Sm D1 (AHRD V1 ***- B6TXH2_MAIZE); contains Interpro domain(s) IPR006649 Like-Sm ribonucleoprotein, eukaryotic and archaea-type, core

429   434 4.25 Solyc08g079630.2.1 1618~1639(cDNA) AT-hook motif nuclear localized protein 1 (AHRD V1 ***- Q8VYJ2_ARATH); contains Interpro domain(s) IPR005175 Protein of unknown function DUF296 siR1001 SIR1001 CDS TCACATGATTATTA AAACATAAT (SEQ ID NO: 435) 218 7.4 8.4

435   436 3.5 AT1577470.1 1437~1460(3′UTR) replication factor C subunit 3

435   437 4.5 Solyc04g055110.2.1 1474~1497(cDNA) Mitochondrial import receptor subunit TOM34 (AHRD V1 *--- TOM34_RAT); contains Interpro domain(s) IPR011990 Tetratricopeptide- like helical Normalized read counts are given in reads per million B. cinerea sRNAs. Reads were summed from individual sRNA libraries for each category: B. cinerea-infected Arabidopsis (“A”), B. cinerea-infected S. lycopersicum (“S”), and cultured B. cinerea (“B”). *AS (aligned score): Target gene alignment was scored as described in Materials and Methods.

TABLE 2  Primers for constructing short tandem target mimic (STTM) against selected B. cinerea sRNAs listed in Table 1 sRNA Primer* Primer sequence Bc- 3.2- GccATTTAAATatggtctaaagaagaagaatACCTACAAG siR3.2 STTMSwa48ntlink-PF ATctaCCACAATGTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 438) 3.2- GccATTTAAATtagaccataacaacaacaacTACATTGTG STTMSwa48ntlink-PR GtagATCTTGTAGGTaagcttgggctgtcctctccaaatg (SEQ ID NO: 439) Bc- 3.1- GccATTTAAATatggtctaaagaagaagaatGCCCACCTA siR3.1 STTMSwa48ntlink-PF CActaAGATCCACAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 440) 3.1- GccATTTAAATtagaccataacaacaacaacTTGTGGATC STTMSwa49ntlink-PR TtagTGTAGGTGGGCaagcttgggctgtcctctccaaatg (SEQ ID NO: 441) Bc-siR5 5- GccATTTAAATatggtctaaagaagaagaatAAGTATACA STTMSwa48ntlink-PF TTctaCCGAGTCAAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 442) 5- GccATTTAAATtagaccataacaacaacaacTTTGACTCG STTMSwa49ntlink-PR GtagAATGTATACTTaagcttgggctgtcctctccaaatg (SEQ ID NO: 443) 3.1-3.2-STTMSwa48ntlink-PF (=3.2-STTMSwa48ntlink-PF) 3.1-3.2-STTMSwa48ntlink-PR (=3.1-STTMSwa48ntlink-PR) 5-3.2-STTMSwa48ntlink-PF (=3.2-STTMSwa48ntlink-PF) 5-3.2-STTMSwa48ntlink-PR (=5-STTMSwa48ntlink-PR) 5-3.1-STTMSwa48ntlink-PF (=3.1-STTMSwa48ntlink-PF) 5-3.1-STTMSwa48ntlink-PR (=5-STTMSwa48ntlink-PR) SiR1 SiR1- GccATTTAAATatggtctaaagaagaagaatCAGAATTCTA STTMSwa48ntlink-PF CTctaCTTGCTTCGAgaattcggtacgctgaaatcaccag (SEQ ID NO: 444) SiR1- GccATTTAAATtagaccataacaacaacaacTCGAAGCAAG STTMSwa49ntlink-PR tagAGTAGAATTCTGaagcttgggctgtcctctccaaatg (SEQ ID NO: 445) siR1010 1010- GccATTTAAATatggtctaaagaagaagaatAGCAATCAAA STTMSwa48ntlink-PF ActaATTCCCCCGAgaattcggtacgctgaaatcaccag (SEQ ID NO: 446) 1010- GccATTTAAATtagaccataacaacaacaacTCGGGGGAAT STTMSwa48ntlink-PR TAGTTTTGATTGCTaagcttgggctgtcctctccaaatg (SEQ ID NO: 447) siR1008 1008- GccATTTAAATatggtctaaagaagaagaatGCATAAACTG STTMSwa48ntlink-PF ATctaCATCATCACAgaattcggtacgctgaaatcaccag (SEQ ID NO: 448) 1008- GccATTTAAATtagaccataacaacaacaacTGTGATGATG STTMSwa48ntlink-PR TAGATCAGTTTATGCaagcttgggctgtcctctccaaatg (SEQ ID NO: 449) siR9 9- GccATTTAAATatggtctaaagaagaagaatTCTAAAAATG STTMSwa48ntlink-PF CTctaCATCATAAAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 450) 9- GccATTTAAATtagaccataacaacaacaacTTTTATGATG STTMSwa48ntlink-PR TAGAGCATTTTTAGAaagcttgggctgtcctctccaaatg (SEQ ID NO: 451) siR10 10- GccATTTAAATatggtctaaagaagaagaatAGCACCCTAC STTMSwa48ntlink-PF ActaACCTAGAAAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 452) 10- GccATTTAAATtagaccataacaacaacaacTTTTCTAGGT STTMSwa48ntlink-PR TAGTGTAGGGTGCTaagcttgggctgtcctctccaaatg (SEQ ID NO: 453) siR18 18- GccATTTAAATatggtctaaagaagaagaatTGATCGACTC STTMSwa48ntlink-PF TctaGTTTTGGCTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 454) 18- GccATTTAAATtagaccataacaacaacaacTAGCCAAAAC STTMSwa48ntlink-PR TAGAGAGTCGATCAaagcttgggctgtcctctccaaatg (SEQ ID NO: 455) siR15 15- GccATTTAAATatggtctaaagaagaagaatTCAAACAACA STTMSwa48ntlink-PF AGctaGTTCAACACAgaattcggtacgctgaaatcaccag (SEQ ID NO: 456) 15- GccATTTAAATtagaccataacaacaacaacTGTGTTGAAC STTMSwa48ntlink-PR TAGCTTGTTGTTTGAaagcttgggctgtcctctccaaatg (SEQ ID NO: 457) siR17 17- GccATTTAAATatggtctaaagaagaagaatCCAGTGCCAT STTMSwa48ntlink-PF TctaCATCATTTTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 458) 17- GccATTTAAATtagaccataacaacaacaacTAAAATGATG STTMSwa48ntlink-PR TAGAATGGCACTGGaagcttgggctgtcctctccaaatg (SEQ ID NO: 459) siR22 22- GccATTTAAATatggtctaaagaagaagaatACTACACCCT STTMSwa48ntlink-PF TctaGACCACGTTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 460) 22- GccATTTAAATtagaccataacaacaacaacTAACGTGGTC STTMSwa48ntlink-PR TAGAAGGGTGTAGTaagcttgggctgtcctctccaaatg (SEQ ID NO: 461) siR24 24- GccATTTAAATatggtctaaagaagaagaatGTCAAACAGA STTMSwa48ntlink-PF GActaGGACCAATCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 462) 24- GccATTTAAATtagaccataacaacaacaacTGATTGGTCC STTMSwa48ntlink-PR TAGTCTCTGTTTGACaagcttgggctgtcctctccaaatg (SEQ ID NO: 463) siR25 25- GccATTTAAATatggtctaaagaagaagaatAAAACCAAAA STTMSwa48ntlink-PF TTctaTGATTCACTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 464) 25- GccATTTAAATtagaccataacaacaacaacTAGTGAATCA STTMSwa48ntlink-PR TAGAATTTTGGTTTTaagcttgggctgtcctctccaaatg (SEQ ID NO: 465) siR1015 1015- GccATTTAAATatggtctaaagaagaagaatACCGATCAGA STTMSwa48ntlink-PF ctaCAACCATCAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 466) 1015- GccATTTAAATtagaccataacaacaacaacTTGATGGTTG STTMSwa48ntlink-PR TAGTCTGATCGGTaagcttgggctgtcctctccaaatg (SEQ ID NO: 467) siR20 20- GccATTTAAATatggtctaaagaagaagaatAATCAGAAAA STTMSwa48ntlink-PF ACctaAAGAACACTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 468) 20- GccATTTAAATtagaccataacaacaacaacTAGTGTTCTT STTMSwa48ntlink-PR TAGGTTTTTCTGATTaagcttgggctgtcctctccaaatg (SEQ ID NO: 469) siR1021 1021- GccATTTAAATatggtctaaagaagaagaatACATGTTTTG STTMSwa48ntlink-PF TTctaCATCACTGTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 470) 1021- GccATTTAAATtagaccataacaacaacaacTACAGTGATG STTMSwa48ntlink-PR TAGAACAAAACATGTaagcttgggctgtcctctccaaatg (SEQ ID NO: 471) siR1002 1002- GccATTTAAATatggtctaaagaagaagaatTGTGTTACAA STTMSwa48ntlink-PF AGActaTTTGAAGAATgaattcggtacgctgaaatcaccag (SEQ ID NO: 472) 1002- GccATTTAAATtagaccataacaacaacaacATTCTTCAAA STTMSwa48ntlink-PR TAGTCTTTGTAACACAaagcttgggctgtcctctccaaatg (SEQ ID NO: 473) siR28 28- GccATTTAAATatggtctaaagaagaagaatAAGAAGATCA STTMSwa48ntlink-PF CActaGTTTCAAAAAgaattcggtacgctgaaatcaccag (SEQ ID NO: 474) 28- GccATTTAAATtagaccataacaacaacaacTTTTTGAAAC STTMSwa48ntlink-PR TAGTGTGATCTTCTTaagcttgggctgtcctctccaaatg (SEQ ID NO: 475) siR31 31- GccATTTAAATatggtctaaagaagaagaatCATTCACGAC STTMSwa48ntlink-PF CctaACAAGACTCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 476) 31- GccATTTAAATtagaccataacaacaacaacTGAGTCTTGT STTMSwa48ntlink-PR TAGGGTCGTGAATGaagcttgggctgtcctctccaaatg (SEQ ID NO: 477) siR29 29- GccATTTAAATatggtctaaagaagaagaatCCCAAAAAGG STTMSwa48ntlink-PF ActaCTATCCAACAgaattcggtacgctgaaatcaccag (SEQ ID NO: 478) 29- GccATTTAAATtagaccataacaacaacaacTGTTGGATAG STTMSwa48ntlink-PR TAGTCCTTTTTGGGaagcttgggctgtcctctccaaatg (SEQ ID NO: 479) siR41 41- GccATTTAAATatggtctaaagaagaagaatTTCTACTCCC STTMSwa48ntlink-PF GctaAAAACTATCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 480) 41- GccATTTAAATtagaccataacaacaacaacTGATAGTTTT STTMSwa48ntlink-PR TAGCGGGAGTAGAAaagcttgggctgtcctctccaaatg (SEQ ID NO: 481) siR35 35- GccATTTAAATatggtctaaagaagaagaatAACGCGACAT STTMSwa48ntlink-PF GctaGCACAGTACAgaattcggtacgctgaaatcaccag (SEQ ID NO: 482) 35- GccATTTAAATtagaccataacaacaacaacTGTACTGTGC STTMSwa48ntlink-PR TAGCATGTCGCGTTaagcttgggctgtcctctccaaatg (SEQ ID NO: 483) siR57 57- GccATTTAAATatggtctaaagaagaagaatCCAACGAACC STTMSwa48ntlink-PF AGctaAGATTATCTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 484) 57- GccATTTAAATtagaccataacaacaacaacCCAACGAACC STTMSwa48ntlink-PR AGctaAGATTATCTAaagcttgggctgtcctctccaaatg (SEQ ID NO: 485) siR43 43- GccATTTAAATatggtctaaagaagaagaatCCCAACAAGA STTMSwa48ntlink-PF GctaAAAGCTCCCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 486) 43- GccATTTAAATtagaccataacaacaacaacTGGGAGCTTT STTMSwa48ntlink-PR TAGCTCTTGTTGGGaagcttgggctgtcctctccaaatg (SEQ ID NO: 487) siR40 40- GccATTTAAATatggtctaaagaagaagaatAACCAATACA STTMSwa48ntlink-PF ActaGCCCATTCCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 488) 40- GccATTTAAATtagaccataacaacaacaacTGGAATGGGC STTMSwa48ntlink-PR TAGTTGTATTGGTTaagcttgggctgtcctctccaaatg (SEQ ID NO: 489) siR48 48- GccATTTAAATatggtctaaagaagaagaatTTGATCGATA STTMSwa48ntlink-PF CctaTGTCACTTCAgaattcggtacgctgaaatcaccag (SEQ ID NO: 490) 48- GccATTTAAATtagaccataacaacaacaacTGAAGTGACA STTMSwa48ntlink-PR TAGGTATCGATCAAaagcttgggctgtcctctccaaatg (SEQ ID NO: 491) siR49 49- GccATTTAAATatggtctaaagaagaagaatTATCAAAAGA STTMSwa48ntlink-PF CctaATAAGCCACAgaattcggtacgctgaaatcaccag (SEQ ID NO: 492) 49- GccATTTAAATtagaccataacaacaacaacTGTGGCTTAT STTMSwa48ntlink-PR TAGGTCTTTTGATAaagcttgggctgtcctctccaaatg (SEQ ID NO: 493) siR58 58- GccATTTAAATatggtctaaagaagaagaatCAGACAATGA STTMSwa48ntlink-PF ActaTCCCAATTTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 494) 58- GccATTTAAATtagaccataacaacaacaacTAAATTGGGA STTMSwa48ntlink-PR TAGTTCATTGTCTGaagcttgggctgtcctctccaaatg (SEQ ID NO: 495) siR1005 1005- GccATTTAAATatggtctaaagaagaagaatTCCTATTGAA STTMSwa48ntlink-PF GctaAAACTCTTTAgaattcggtacgctgaaatcaccag (SEQ ID NO: 496) 1005- GccATTTAAATtagaccataacaacaacaacTAAAGAGTTT STTMSwa48ntlink-PR TAGCTTCAATAGGAaagcttgggctgtcctctccaaatg (SEQ ID NO: 497) *Forward primers are denoted as “PF.” Reverse primers are denoted as “PR.”

TABLE 3 Predicted B. cinerea sRNA targets in V. vinifera SEQ ID Target site sRNA and target in V. vinifera Alignment NO: Molecular function position Bc-siR3.2 VIT_10s0092g00240 Target   Bc-siR3.2

498    24 carbohydrate binding, hydrolase activity carbohydrate metabolic process CDS + UTR Bc-siR3.1 VIT_12s0028g01140 Target   Bc-siR3.1

499  30 Pentatricopeptide repeat INTRON VIT_06s0009g01890 Target   Bc-siR3.1

500  30 exonuclease intron VIT_10s0116g00190 Target   Bc-siR3.1

501  30 KNOX1,2 domain containing protein Intron Bc-siR5 VIT_05s0020g01790 target   Bc-siR5

502  36 Lipase CDS VIT_01s0011g01000 target   Bc-siR5

503  36 NB-ARC and LRR domain intron VIT_05s0077g01510 target   Bc-siR5

504  36 DUF7 domain intron

Example 2 Sequences of Promoters and sRNA Resistant Targets

A. thaliana (At) BIK1 Promoter: SEQ ID NO: 1 attttattatattatatagcgatgagagagacagagcttgaaggttctttttagcgaaagagaaaaatccaggaaga taggcgaaaaggaagatgaagcgaagatgaggttaatataatactcatgttaaatgacaaaaatgcccttatatgat taatgatattaccatttgagcttgctgtggaagctgtaacgaaccgaaaattaaaaacagaataacgaacatagacg gagaatatgatattattcgttttaccaaagaaactaacaaatagttttaactttatctaacaaaggggtaaaacggg taatttgtttgggatgaggtggagcgtagcggacaatcgagaaattaaaagtttggcttggggacgaagttaaaggt gggctttaacgttttaaattggctgactcggacgatatttcttgtatttaataccaaaaatgaatgactttataatt catttgtagattgaaagttacgtattgattcgaaaatcaacacattgtgttttcaagtgggcataaactataacacc ttgttgattgattaatagattacctaaagacattatggtttattactggtctttcaatatatttttatcgcattgtc aatgatattgtttttgtatcccaagtccactgttttggtctctacattcattttgattgggatttatctttttaaaa tttcttctaatgttttttcgatatggttattacttgctttgattttcttttcagtatgtgtattgctttgcaaattg tttttttcttaagatgaaaaacaactcattaaattgtttgagaaatactactaaaacaaataaacaatgaggagaat tatggaaaacaaagtgtaataggctttaattcattgctagtgggctttttgggcctatgggcatattacttaccact atccaacccaaaatgccaaataaccgacatgtctcaccaatccaattttgggccatacggccgaaattatttaaacc tgtgctcataatttactttacaaattattacttttccataaattgtggaaaagttatctgtaacatccgattcaact ggagtctagactactatagacattgatacgttttgagtttttagatacttggaagatatatgcatttatgaatacag attacagacacatactagtactactgtatgtctgtatatggatacaaaaaaaatcatgtatgaatactaaaatttta ttagaaatctatttttcaattgttgcaacaatcaagttgtcaaatttatttttgtaaccgttaaacaaacaaatatc gatttaggtttctaatctgaattgacatctcaaacaaaaaaggctgaatactttctgaaaatagtgtatggaatgaa ggtggcttttagagccattataaccggaagaaaattcaggtgacttttagaaccattataaccggaagaaaaggtga attttaatttttagctgtgtggaagacacggcaagtccaagtagtaccttcgtacgtcaatattgtccaaccggccg tgtcgaaaatcttcttgagaaaaattggattttcatctataaaaaaaaaaagtccaagtaataccaaacaaagacag cgacgtgtaaaacaatacaagactcataatcacaaacctaccacccaagtcaaacctatattccatttagtgaattc ttgattatgacttcttgaaatcatttgtattcatatgtataattatttaagtcatttttctgtaagtaaaattttta tatatctagaataacgagttc cctacgacaagatacagttgaacgtaaatgtgacatctcaattttcattggtgtctagtactctagtgattaggttt tcgacatttattgtactgattaagtaaaaattcatggtacaaacatcgaatatatatttttctgcttacacacacca attaacgtggatagaccaattgaaatattttgttacgacaaagcaaaacaaaacaaacgtcatgtttcgctgtttgt ttgtcgtcccgttaatggtaatctttcagacacatacagtacccaaacaagtaatttgactaaaattttctctctgt ctaaatttcagaagaaaaaaaaactttaggatatattgccaaaagatcttaaaaatgggtcatatcattttgatcat atagaatccaacgacctttatcttttcgccgaactatacttttttgtgtccatttgtttgactttctttcacacaca catccacaaagaaaaaaggaccattcttctccttcttctagtcacccctcgtgcctctctttaacaccaaacccaaa actcccttctctttcttccttcctctccgatctccgttcacatctctctctcatctttatcttcttctttttttgcc ttgtgggttgaaagtttctatattttctctttctcttctgtttacataatccattttcagctcaagcagctgaagaa taacgatcaagaaccaaaaaagaagaaaacgaatctgttcttagctttg At PDF1.2 Promoter SEQ ID NO: 2 ACGACGTTGGACTGTTTCATCATATCCCATAAAAATACATGATTGGGGTGAAAATCTTGA ACATATTAAAAAAATATTAAATCAAAATGATAAAGATAGGGATTTATAAATGTAAAACGG GCGTGTCGAGAATTTTATGGACATTGGGACAAGCTTTATATGCAGCATGCATCGCCGCAT CGATATCCCGAGGTGCATCGTTTCTACTTTCATGTCCAAATTTGGGGTTAACTCACAATA TATATCATGTTGCCTATGTAAATTTATAATCATAAATCTAAACCCAAATTTTAATCCTCA TTCCAAAGCAAAAGTTCTAAGCCCTACAAAAATATGTATTTCCCAAGTTTAAAAAGAATT AATCTATACTTTTACAAATTTAAATTCTGATCTCTTATAATGTTCGGTTTTTCCTTTTTT ATTTATTAAGTTAGTTAAAATTTGCAGTTATTTTGTTGAATGTCGTTGTTTACGAATTTA CGAATAATACCTTTATAGCTAATCTACAAAATTTTGATGACTGACAACACCGTTAATGTT TTTTTTTAAATTACCCTGAGCCTCTCACTTGCGGTCAGACCATGCATGTCGATAGTCCAT TACGTTTAAGGCCACAATCAACTATAGTTTGTTTATCAATAGCCAACTAAGCTAACTTTT AGGTTCCTGCCCTCTCCGTTCCTCCGGTACCAATCGTTTCTTTGTCCCTTCGATAGTTTG AAAACCTACCGACGGTGAGAGCAAAATATTGATGAATCATCCAATTTTCAGTAATAGGTG TGTCCCAGGGATATATAAATGGCGAAACTACGCGAGAACGGTTCCTTGTTCTGCAAACTT GGCGGAACAATGCTGCTCTTGAGATCAACCAAACCATATGTTTAGTCCACAACGATCTAT ATGTCTAGGGGTGATCCTCTAATCGAAAAATGTTGTATTTGTTCGACGATGACGAAGGTC AGACTATGAACTGCACAGTCTGCACTTGTCCTAACCGCGAGAATCTCTGACATCAATATA CTTGTGTAACTATGGCTTGGTTAAGATATTATTTTCTTGAGTCTTAATCCATTCAGATTA ACCAGCCGCCCATGTGAACGATGTAGCATTAGCTAAAAGCCGAAGCAGCCGCTTAGGTTA CTTTAGATATCGACAGAGAAATATATGTGGTGGAGAAACCAGCCATCAACAAACAAAAAG CAAGATCTTATCTTTTGATATTGGCTACGGGAAGATGATGTCTGTTTAATGTGTGGGGTT ACCACGTTATTGTACGATGCACAAGTAGAAGATTAACCCACTACCATTTCATTATAAATA GACGTTGATCTTTGGCTTATTTCTTCACACAACACATACATCTATACATTGAAAACAAAA TAGTAATAATCATC At BIK1 homologous gene in tomato (TPK1b) Promoter SEQ ID NO: 3 TTGCGTTTAATTTGTATGAATGTCATTTAATTTTTAGGATCGGCTTAAATTTGAAATTAA AAAAGCAAAATAATAATACTAGTATTTTCTAACTTTGTATTTTAATGCATGACATTATTT TTAGAAAAAATTGTAACGAAGAGAATCATATTTATGATAGAATTATTTGTAATTACTATT TGACTGATATTACTAGTTTAATTATTTCGCACACAAAGTATATTTTTTTAAAAAAAAATA TTTTACATTGATTATTTTCTCTCTATCCCAACACCCCATCCCGTCTTTATTTTTATAGTA TTTATTATACAAATATTTTAAAAGTATCTTATTGAACATCAAAATAATCTTTTTTAAAAA TTATTTATATCCCCAAAAAAATTATATGCACGTGTGAAAATGAGAAAATGTTGGTTGGGT GTGAATAATTTGTTGGTTCCCAAATATGATTATAATCCAAGAAAATTGGAAATTTGATTA TTGCTTCCTTTTGACTTAAAACTCTTTGCTAAATTGCTAAGCATTCTTTTTAATTTTGTT TTTCCATTAATAACAATTTGGGTAATTCATATCCACTAGTCGGTGGATTTAATAGAAGTG ATACATATTTTTTTGATGTTATTGTTAATTAATAGTGAAAGGTCCTTTTTTCTCTCTCCT AATTTATATATAATTCATTTTTTAAAATCAATTTTGAAAGAATGATATAGTTTCTATATT TAAGTAATGATTTATTTTATTGATAATAAAATAAGTTATAATCATATATATATTTTTAAT ATATTTAAAATTATAATTTAAATTATTTATATCACATCAATTGAAACGGATGAAATTATT TATTTTAAAAAAAATGATGAATGGGTGGCATCCATAAAAATGTGACATTTCTCCATGTGT TTTGCTTAAATGAGATTTTTGACTATTTTTCTTGTGTTCATATTTATGAAGAAGATCAAC AATAAATTTTTATCAATAAAGAGGAAATTAAAAGTTGATTAATATTAAAAATCACAAATA TTTATTGAAAGTGAATAAATTTATAGTTATTACACATATATGGAGAGAGATCAAAATCAA TATGCTAATTTTTTGTAATGGAAGGGCACAATGAAAATAAAGTTAATTTTCATGACTAAT TTAATCCATATAGTTAAATTCTAATCATATAAATTTCAGTGAATAAGTTCATTTGATTTT TTTTAGATCTAATATTAATTATTAAGATGTAAATGTTAACTATGTTTTTATTAATGTTTC AATCACTGTGTCTATATTTGAATGATTACTACTTGTAATTAAGTGAAAAAATTCAGTATT TTGTGTATTAAAATTTTTTATTATTGAAAGAGATATAGATTTAAGTGGAAAGTTAATAAA GAAAATTGCAGTTCGCCCTCAAATGAATTATCTTTAAAATTTGTTTAATAATATTTGGAT CAATAAGTTAACGGAGTGGAGATTTTTAAAAGATGATAGTTAAAATTTGCACATAACCGA ACAAATTGTCTATTTAGGTATGTAATTTAGAGAGTGTCTCTTTTGAGGTTTGATGTTTAG GGTTCAAAAATTGTCCGTTTTGGTGCCAGAAACGTGCCTACAACCACCATCCAATCCATT CTCAATCACAATCACCATCACTGACACCCAATCACTACAATAAGTCGTCATTGCCGCCAT CCTTATAACAAAAGTAATTTTTTTGCAGTCATAACTATATACTTTAATAAAAAAATGTAA ATTTTTATCGACATTACTTAAGTATCATTAAATTTACTATCGCTAAAATCTTTAGGGAAA TTTATAAAGAGTGTTAATTGTTATTAAAAAAATTATATTTATCGATAATTAAATTATTGT TGCTAATTACTTACCATTGACGACCATTTTCAATGTAGTACATCCAATATTATCGCAATA AATCATTATCACCCGTCATTACTATTAATTACTACTTATATATCGTCAATCACCATCATC ATTAACCATTGCTCTTCATCCACCATAGTTATTGTCTTTCAAGCATTACCATCATTCATC ATCATTATTAACTACTTATATATCATCAATAACGATTTATCATTCATCACAATTATTATT TATCAACATCACCTATCGCTCTTGATCATTACTATTAATCATCATTAACCTTTAACTGCA ACTTACACTATTGTTCTTAATCGATATTCACAATCACCATAGTTAGTCATCACCATGAGT CCTAGCCACAAATTCAAAGCAAAACACCCTTAAAGCCTGGTAGTGTGTGTGAATTAAAGA CCAGCAGTCCAAAGAGAGAGAGAGAGAGAGAAAATGTAGACTTTAAAGATATGTAGTAGG ACCAGTCTGCCATTAATATCTCCTTCTACCAACCTTCCTCTCCTCTTTCACTACCCTACA TTTAACATTTTCCTATAACCACTGCTTTAGATAAGTCAAATTTAGCTCTTTGTTTTGATC TCTGTTTCAAAAGAAAACACCTATTAAGCAGCCATCATCTTTCTTATCTTTTCCAAAACC AAAACTACTGACTTTTCTTGAAAAAAGAAGAGGTGGGGTGCTTTCTTTTCTTCAAAAACC TTCTCTTTGTTCTTGAAAAAACAGGACTCATTCATTTTTTTTTGTGTGTTTCTTTCAGAA GAAATAACAAAGACCCTTTCTCTGTTTTCTTCATATTTCAGCTTTGAGCTACTTGGATCT GTTTTTTTTTTTTGAATATACAAGTAGTTTGTGTGTTCTGGGGTCTACAGAAGAAGGAGA AGCTAAAAGGGGTGATTTTGTTTTTTGTTTGTTGTTGTTCTA AtML1 promoter (AT4G21750.1 promoter sequence) SEQ ID NO: 14 aagcttatcaaagaaaaaacaagaacaaaacgatgcatagtttctaaaatgtgctaaaattcagaaactgaaacatgattcattgtctgaaactt tgtttcaaattactgaaaataatcattcactggaccaaaacaaataaataaaataaaatcgaatttctgaatttggaaattggtttttggttttt aattttaaacaaaacaaaaacgaaatttgaaggcaataaatgagttagttggtaggcagaagtcactcgttcccactagctattattattagaag aaacgtccccacaactccaaggcgtttcagttcctttaatttactgaattaccctcctcatatctataaaaaatcacctcttgtaccaatgcccc atttacacatcctgtcgtttatttctagactaagtggactacatgtcggttatttgattcgcaccatgcgtatttggattatcgctaacacaccc cttcaaacaatacgcttaactcgtattacaaaatttcaagtgatgaattatctatgtataagatatagataggaacaactaagcatcgagaaatt tgtatataaatcaactagacttatatatatttcgatacagaatttatacgtattatatcaaattaattagtaattgtttcctctacgtgagttta attaacaatgataagctacattgagtgtatcagttctaaaactttatagtatgctacaatcaatttttctaagtaacaacttcaagcaaggaatc acacacacacagtggtacataataaacttgattttaatatcatatgatcagcatcattaacggaataagttaagtaattcgtcatccatactact aagtcatattaaaatcataatcaaacttaaaagccgattagaaagagagcaaatatatctaaaaattcacgaggaagacgacaaatgcaaggaaa cacagctagtattattaaacttaatagatattggatgaatgactgcataatatatatcacattaaaagtggacataaatttgcatatgtgtaatg tacctctccacaattaatcgcggaccatttattttactattacaagtcaagtaactttatattgttgatccataattcttttcgaacataaaatc atatacttaggccattttcaactgtcaaaactcgaatccgagaaccaaatttcaccattttccaaaaatgatgagtgtcgaccaaatggggtact actgtctaatcaggaacttgtgaacaaattttcaaccttttccaaataagacgagtgtcaaccaactttttccaaccaagagatattgggttgct acacaaatacttaatagccattgcatatttatgcatatgcaaatgcagggtcgtggcgtcagaaagaaacataggaccctcaacatatttaatat tttgggagctatatttgactatttcatattagaaaataataataaaaaagtgttggttttatatcaaattgtaatttacgaaaaacttatgcttt tgcgcaatgatttttgtaaagtatctactatgtttagtgtttacattgattagtaggctgccgttttttttcttgtgtattatgtactatatatg aatatgaacatttgtaaaagtgaatcttgtcattttcttgttgaaaacatatatagtatgtgcaaacaaagcataggttaatccaataccacaca aataacacgtcaggtaaatccaataataaatcgtatgtgcatgtatgtgtattcatgtatgttacatgaatgtctgaatcagtcagtgtacgtat atgatgtaggtgatgtaaatcttaatgtatgagctgtttcttggaccatggtccacaatggatattgctccccaactacattagtcaatcgactg gccaatttttaattaagataattaatccaaactaccattaaatataactttgaccttttttctattcatttttagatattattggaacttacgta gtttacatgcatctcatccctttcttttgctccttgaaagtgggtccaatcacaaaaaatgatcttatattttgtattttgtattttaaaaactc ataattatataggttcaaaaatttaattaacatcagtgtatactataattactactctagccaacaagataaattcattttgacatcagccaaaa gataaaaatttggttaaaaactattggattagcttttagtatttaatattttatgtactgattaaatacgaatttagaaatctaggatataagtg agggtgtataataagggaggggtggaccattaatagcgatgtgcaattaaaaattatgattaagaatctaggaaatttgtagattgcttagttat ttttatggcgatcgtcgtgtcaatgtcatggattttgaaactttaaattaatctcttaaattagcacctacctttgaattttatagaatcttttt attttatatgtttaattttatagaatctaactagcttattttgagattaaattgtttagttacttttataacagtataaatgtataatgaggacc taagaatgtagtcctgtaatgttcttgctattctacttaatctcatcaccaatcaaccatcaaaagaagctagtactaataaaacctgcaggtat tcgaataataattaagctcaaacactatactaatttatggaggattatatattcaatgaattaggaacctcatgatggacattattgactgatat aatgtgtatactaattgtgagtatttaaaaaccatacaaagcatttatatgtccacatatattggacacacatgcaatcaatgttcaatatgctc cacacacagaaataaaaatactctttctgatcatatgatacatcatacatatactaaaaaaatctaaaatgaactataaccacaagcatatataa taacaatgaaatggtaatgtttcttcatttttatttgttcaaattcttattcggttgttttttcttaccctacgagaatccgtgaggtcaaaggg aaacagtgattttttttttgtattttgttttttaaattgatgaactgtaaaactctctctctagaaaaatatataagtagtagtatgaattttct ctcactaaaagcattaatggacctttcgataatcataaatgcaatgcaccctctctatgcatttcgcaataactccttttccttctgccacatcc tcttcctcacctctttctcttcttccctttctcctaagttcctcctccaccaaattctccatttatttcgttaactatcctccatttgttttctt ctgaagagtgatatattctacctttctctggttaaagaaactccctgaatccaccggttatgtcttgaccggctataagcctataaactgatgcc ctaagacacctttttaggtttctcaataattctccgcatctatcttttcttctccacaagtaagagaaccagaaaaccagagaagaagccgagct agctagggtttcattgtgtgcacaaaagtaagatctctctctctaaccaatacttgtgtaatttgtctttgtttctttgagcaaatattgcatgt ttgttcatattagccggatccgttttatattttttcatgatctacattttatcfflattttgtttgtaaattaatgagttttttttttttttttc tgtttttgtcacgatctaaaaaacaagcgttacaaagaagaagaaaaacctttttggagttagaagtgtaaaaggggtttcagtttgacgaattt tccttagtagttgtgtaaaaaaaggccattgacttaatgtcaactctatatatctacacatttttttattaattagtttttgtttttttcccact tcatttacctttagtcaatgaatttttactgaaaacgttttttcaaggtcaatttcactgagttaaaaaaaaaagttttatttttaaccaaaaat tacgttttttcctaggcttcggtaacctgtgaattcctctatctcactagcttttatgtagaagagagagaaggcaacattaaattcgatctaaa acttcaagaaaccaaaacaacacttcaaaaaaaaaaagagatctgttctatagagttttaatcttttctttcgactcgagtttggctcaacaaap tatatcgatttggcactctaaaatgtaagtagaaccaaatgaatcttgtattttatgtacgttaataaaaaattagggtttcctagacgacaatc tcgtcatccgtttcttctttgtctacctctgcgttttcttgtagatccgatgatgtgctcagtcttgtgactttcaagattgattttatcgttat tgtttgaagatatgtggtttgattattttctcaacacattgtgtccttttagcgctttacttcagtttctctctaattttcataatattattatt gaacattatgcttaattattcatccgaatattcgtgtcccattttttaaattgaatttcaggataacttgtattttatatgcaacgaggttatgt cacgtagtgggtgcatttatattcataccctttttgataagatgaatgcatatgcttatataagcgtataggtataaataaccatcaaaaataga gaaaaagaccaatattttgcttttcggttacttatgaaatgtgaaaaagaccatataaatatatctattaaagggaagtatagtttcataaaatc ttgaggattacattccataaaccaagattaccttccgtttttgctttgatcctcttcttatcaaatatataaacatgaccatttgatctttcatt ttggatagtgggatatacaggcagaagaaaatcgagataaatcaactaaatgatttggataatcatcttgaagatttgaaggaaaatccaagagc ttcaaaaactccaaaaattgataggcatccatcatcatc Tomato ML1 Solyc10g005330.2.1 promoter sequence SEQ ID NO: 15 ATTTTGACACACGAAAAAGTAGTACGAATATTGAACTCATGATAACTTTATCAGTTACTTCA AGACTCTCATTTTAACACAAGAAATATATTTTACAAAGAAAAAGGGAACATATTTTACAAAG CTTTATTTTGTATTTTCATTAATAATTATTTTCAAGGCTTGAACTCATAATAATTTTATCAGTT TTTTCAAGATTTTCATTTTAACACACGAAAAAGTAATATGAATATTGAACTCATGATAATTTT ATCAGTTACTTTAAGACACTTATTTTGACACACGAAAAAAGTAATACGAATATCAAACACCG AATACGAAAGAAAAAAAGAAATGAAAGCATTATAGTAGTTGCCAACCGCCCCTTCCTCCTC CTCTCTCTCTTCAACAACAACATTAACACCTCTATAGCAAGTCATAAATGCTATTTCATCCTC TCTATACCCTTTGCATTAACTCCTTTGCTTCCACAATCTCTTCTCCCACCTCTTCACCTTCCCC TTTTCACACTTTCTTTCTCTTTCTTTTTTTCTTTCATCCTTAGCCTCAAAACTATTCTTCTTAAA TTCTAGTCACAAGAAAAGTGTTCAATTTCAACCTAGCTTCACTAAAATATATACATGTTCATT CTCCAAAAAGTACTTCTTGTCAAAACTTAGATTTAACCATTTTCTCAAAAACCCTAATAACAT CAACAACAAAAAAGAAGAAGAAGGTGTGTTCTTGCTTTTGTCACAAGGCTTCTCTACAACTC ATGTAAGTCAAACATATACTATCATCTTCTTGAATTTGTTGAATTCTTTTTTACTAGCTTATAA GTGTACTATATTGTTCGAATTTTCTAAAAATATTATCCGATCTTTTAGGAACAATATATATTT TTAAAGATCCAATACAAATATAACATTAGTTTCACAGAGTCCGAGCAAAATAGATAAATAGT TGTAAATTCACTTGTATTTGACTTACCTTTTCATTTTTCCGTTATATTTTGCAGAAATAGAAAT GCCAGTGAAGTTGGACTCTGCCTAGATACTCGTGGACGTTATATCATATACAAGTACCTAAG TTTTGAAAAAAAAATTAACAGTGAAAAAATATTAGTTTTTGAGTTCACACTATGTCAACTCT ATCTTTGTTTTTTGCTAAATTTTTCTAGTTTCAAGTCTTTTTTTTTGTTTGACTTGTAAAACTTT TTTCTTTTACATTATTTTTATCCCCTTAGAGATTCTATAAAAACTCTATGCCCTAACAAAATTT CTTACTAAACAAACAGATATATCAACATATATAGAAACAAAGGAGAGAGAAATTGTTTCTA TGGCTTGAAGGGCTTATGTCATATATGTTATATATGGTGTAAACTCCATCACTATGAAGTTTC TGGCAAGCGGTGAATTTCATCGTAGGTAATAGGAGGTAACAGGTATTCAGTAAGTCGTAATT TTAACATCGAATGTTTATACGAATCATTTTTATACAATAGATGTGAGTTCAATTCTCTCTGTT ATTCTTTGTCTAGAGAGTAGTAAAAAAAAAGATAAAAAGATCCGTTCGTTCTCATCTCTCTC CAATTGTTGAGATCTGTTTGGATCTTGAGTTATTAGGTACTAATAAAGACCTTTCAAGTTGAA TTATTCAATTTTATTATTATTTTTGCACTTTTGGACATCATTTTATGTTTTTAATCATGTCATA ATTATATATGCATGTAGATGAAATAAATCAAAAAGTAGATTTTTATTCAAGAATCAAATAAT TTCTTTATGTTTTTTTTCTTAAATTTATCTTCTTTTGCTTTTTTTAGGGGCAGATTAAAA

Example 3 Sequences of sRNA Targets and Mutations for Making sRNA Resistant Targets

Polynucleotide sequences for sRNA targets (MPK1, MPK2, WAK, PRXIIF, MAPKKK4, S1 F-box (Solyc03g061650.1.1), Autophagy-related protein 2 (Solyc01g108160.2.1), S1 Vacuolar protein-sorting (Solyc09g014790.2.1), S1 Pentatricopeptide (Solyc03g112190.2.1), and TOM34 (Solyc07g066530.2.1)) are provided. Underlined sequences represent target sequences for sRNAs. Alignments of sRNAs to wild-type target sequences and mutated target sequences (target site synonymous mutations) are also provided.

SEQ ID NO: 4-Bc-siR3.2 Target At-MPK1 GTCAACTGTCCGAGCGTTGGCCAAATCTCTCTCACTTCCACAGGTTTCTCTCTCCGGCCAAAT CTAACCTCCGGGGAACGTCGTTGGTCACTTATCACCGAGGGAAAACAAAAAATGGCGACTTT GGTTGATCCTCCTAATGGGATAAGGAATGAAGGGAAGCATTACTTCTCAATGTGGCAAACTC TGTTCGAGATCGACACTAAGTACATGCCTATCAAGCCTATTGGTCGTGGAGCTTACGGTGTT GTCTGCTCCTCTGTTAACAGTGACACCAACGAGAAAGTTGCTATCAAGAAGATTCACAATGT TTATGAGAATAGGATCGATGCGTTGAGGACTCTTCGGGAGCTCAAGCTTCTACGCCATCTTC GACATGAGAATGTCATTGCTTTGAAAGATGTCATGATGCCAATTCATAAGATGAGCTTCAAG GATGTTTATCTTGTTTATGAGCTCATGGACACTGATCTCCACCAGATTATCAAGTCTTCTCAA GTTCTTAGTAACGATCATTGCCAATACTTCTTGTTCCAGTTGCTTCGAGGGCTCAAGTATATT

CGATTTAAAGATATGCGATTTTGGACTAGCGCGTGCGAGCAACACCAAGGGTCAGTTCATGA CTGAATATGTTGTGACTCGTTGGTACCGAGCCCCAGAGCTTCTCCTCTGTTGTGACAACTATG GAACATCCATTGATGTTTGGTCTGTTGGTTGCATTTTCGCCGAGCTTCTTGGTAGGAAACCGA

AGAGAAGAAGATCTTGAGTTCATAGATAACCCGAAAGCTAAAAGATACATTAGATCACTTC CGTACTCACCTGGGATGTCTTTATCCAGACTTTACCCGGGCGCTCATGTTTTGGCCATCGACC TTCTGCAGAAAATGCTTGTTTTTGATCCGTCAAAGAGGATTAGTGTCTCTGAAGCACTCCAG CATCCATACATGGCGCCTCTATATGACCCGAATGCAAACCCTCCTGCTCAAGTTCCTATCGAT CTCGATGTAGATGAGGATTTGAGAGAGGAGATGATAAGAGAAATGATGTGGAATGAGATGC TTCACTACCATCCACAAGCTTCAACCTTAAACACTGAGCTCTGAGCTCAAGTCTTGTTTGTAC GGGTAATTTACAGAAAACTTCTTCTTCTTATGTCTGATTGTCATCATAGACTCATAGTGTATA TAGTCTTGAAAAATAAGATGAAGACTAACTTATAGTTTAAGCGAATAGTGATGCCATGGAA GCTCTGTTTTATTTAATTACAAGCTTGATGTGTGTCTGTAACATATGTACATAGAGAGAGCTG TTTTTTTTTTTTAATTACAAGTTTGATGTGTGTCTGTAACATATGTACATAGAAAGAGCTGTG TTTTTTTTTTAATTACAAGCTTGATGTGTGTCTGTAACATATGTTCATAGAGAGAGCTGTGTT TCTGTTTCTCTGTTTGTTTGTTGCGTTCTTGCAGAACTTTTAACCCTCTCATGCAATCCAAGCC TTTTGATG Alignments of sRNA sequence Bc-siR3.2 to wild-type (WT) At-MPK1 target and mutated (MU) At-MPK1 target

SEQ ID NO: 5 - Bc-siR3.2 Target At-MPK2 ATGGCGACTCCTGTTGATCCACCTAATGGAATTAGGAATCAAGGGAAGCATTACTTCTCAAT GTGGCAAACACTTTTCGAGATCGATACCAAATACGTGCCTATCAAACCGATAGGCCGAGGC GCGTACGGTGTGGTTTGCTCTTCGGTTAACAGAGAGAGTAATGAGAGAGTGGCGATCAAGA AGATCCACAATGTGTTTGAGAATAGGATTGATGCGTTGAGGACTCTTAGGGAGCTCAAGCTTC TACGTCATCTTCGACATGAGAATGTGGTTGCTCTTAAAGATGTAATGATGGCTAATCATAAGA GAAGCTTTAAGGATGTTTATCTTGTTTATGAGCTTATGGATACTGATCTTCATCAGATTATTA AGTCTTCTCAAGTTCTAAGTAATGACCATTGCCAATACTTCTTGTTCCAGTTGCTTCGAGGGC TCAAGTATATTCATTCAGCAAACATTCTCCATCGGGATCTGAAACCCGGTAACCTCCTTGTG AATGCAAACTGCGACTTAAAGATATGTGACTTTGGGCTAGCGAGGACGAGCAACACCAAAG GTCAGTTCATGACTGAATATGTTGTGACTAGATGGTACCGAGCACCAGAGCTACTCCTCTGT TGTGACAACTATGGAACCTCCATTGATGTCTGGTCAGTCGGTTGCATATTCGCCGAGCTTCTT GGAAGAAAACCAGTATTCCCGGGAACAGAATGTCTAAACCAGATTAAACTCATCATTAACA TTTTGGGTAGCCAGAGAGAGGAAGATCTCGAGTTTATAGATAACCCAAAAGCCAAAAGATA CATAGAATCACTCCCTTACTCACCAGGGATATCATTCTCTCGTCTTTACCCGGGTGCAAATGT TTTAGCCATTGATCTGCTTCAGAAAATGCTCGTTCTTGACCCTTCGAAAAGGATTAGTGTCAC GGAAGCGCTTCAACATCCTTACATGGCGCCTTTATATGACCCGAGTGCAAATCCTCCTGCTC AAGTTCCTATTGATCTCGATGTAGATGAAGACGAGGATTTGGGAGCAGAGATGATAAGAGA ATTAATGTGGAAGGAAATGATTCATTATCATCCAGAAGCTGCTACCATAAACAACAATGAG GTCTCTGAGTTTTGA Alignments of sRNA sequence Bc-siR3.2 to wild-type (WT) At-MPK2 target and mutated (MU) At-MPK2 target

SEQ ID NO: 6 - Bc-siRS Target-WAK ATGAAAATCTTGATCTTGATTCTATCCTTTGTGACACTCTTTGAGATTTGCGTTGTGGACGC ATGTCGATCATACTGTGGAAACATAACCGTTGATTATCCGTTTGGGATCCGAAACGGATGT GGGCATCCAGGGTATAGAGATCTATTGTTTTGTATGAACGATGTGTTGATGTTTCACATAAG TTCAGGTTCTTATAGAGTTTTGGACATCGATTACGCATATCAGTCCATAACACTGCATGATC CTCACATGTCGAACTGCGAAACCATCGTGCTCGGTGGCAAAGGCAATGGCTTTGAAGCTGA GGATTGGAGAACTCCATATTTCAATCCTACCTCAGATAATGTCTTTATGTTGATCGGATGTT CTCCTAAATCTCCTATATTTCAAGGCTTCCCGGAAAAGAAAGTGCCGTGCCGCAACATCTCT GGAATGAGCTGCGAAGAATACATGTCATGTCCAGCTTGGGACATGGTCGGATACAGACAAC CGGGTATACATTCCGGGTCAGGTCCACCCATGTGTTGTGGGGTCGGGTTCGAATCCGTAAA AGCGATTAATCTAAGTAAGTTGGAGTGTGAAGGATACAGTAGTGCGTATAATCTAGCACCC TTGAAACTTAGAGGACCCTCTGATTGGGCTTATGGGATACGTGTTAAGTATGAACTCCAAG GAAGTGATGCGTTTTGTCGTGCGTGTGTTGCAACTTCTGGGACTTGTGGCTATGAACCTGCT GATGGTGGAGGGCTTAGACATGTTTGCATGTGTGACAACCATAATTCCACTACAAACTGTG ATTCAGTTATATCACCAACCGGTGCATCATCAAGTGTTCGACCAAAAGCTATCGGATCACT GATCATCTACTTCATAGCTATGAACATAGGCTTTCAGAGAAGACAGCGATGA Alignments of sRNA sequence Bc-siRS to wild-type WAK target (WAK) and mutated WAK (WAK-m) target

SEQ ID NO: 7 - Bc-siR3.1 Target AtPRXIIF ATGGCGATGTCAATTCTAAAGCTAAGAAATTTATCGGCACTAAGATCGGCGGCAAATAGTG CCCGGATCGGAGTTTCATCGAGGGGTTTCTCAAAGCTCGCGGAAGGCACTGACATAACCTC GGCGGCGCCTGGCGTTTCTCTCCAGAAAGCTCGCAGCTGGGACGAAGGTGTTTCCTCCAAA TTCTCCACCACGCCATTGTCAGATATCTTCAAGGGGAAGAAAGTCGTCATCTTTGGTCTTCC TGGGGCTTACACGGGAGTTTGTTCACAGCAGCATGTGCCTAGCTACAAGAGCCACATTGAT AAGTTTAAAGCCAAAGGCATTGATTCTGTCATCTGTGTCTCTGTTAATGATCCCTTTGCTAT CAATGGTTGGGCAGAGAAGCTTGGTGCCAAAGATGCAATTGAGTTTTATGGAGATTTTGAT GGGAAATTTCACAAAAGCTTGGGGCTAGACAAGGATCTCTCTGCTGCATTGCTCGGGCCAC GGTCTGAGAGATGGTCGGCTTATGTAGAAGACGGGAAGGTTAAGGCGGTGAATGTGGAAG AAGCACCGTCTGACTTCAAGGTTACAGGGGCAGAAGTCATCTTAGGACAGATCTAA Alignments of sRNA sequence Bc-siR3.1 to wild-type AtPRXIIF target and mutated AtPRXIIF (MU) target

SEQ ID NO: 8 - B-siR3.2 target in tomato: MAPKKK4 Solyc08g081210.2.1 ATGCGTTCATGGTGGGGGAAGTCTTCATCTAAGGATGTAAGGAGGAAATCCACTAAGGAGA GTTTCATTGACATAATAAATCGGAAACTGAAGATTTTCACCACGGAAAAATCAAGTGGTAAA TCTGGATCATCTCGAAGACGACGTAAAGATACAAATTCAGTGAAGGGTTCTCAATCAAGGGT TTCAAGGTCACCATCACCATCTACTGGATCCATAATATTAGTGACCGGTGAAGTCTCCGAGC CATCATTGACTTTGCCTCTTCCCATGCCCAGGCATCTTCCACATGGACCAACTGCTGCAGGAG TTGACAGGGACTTACCAACTGCTTCTGTTTCTTGTGACAGCTCCAGTGACAGTGATGATCTTA CTGACTCACGATTTCTAAGTCCCCAAACATCTGATTATGAAAACGGGAGCAGAACTGCCTTG AATAGTCCTTCCAGTTTGAAGCAGAAGGTTCAGTCCCCTATTGCATCCAATGCAAGCTCAGG AGAGATGCTGAAGTCAGCTACTCTTTTGTCAGACAATCAGGCGATCCCTACATCTCCTAGAC AGAGGCTTTTAAGATCTCATGTACCACCAGGCTTACAGATTCCTCATCATGGCGCTTCCTACA GTGCTCCTGACAGCTCGATGTCAAGTCCTTCAAGAAGTCCCATGAGGGTATTTGGGCATGAA ACGGTCATGAACCCTGGTTTCTGGCTAGGGAAGCCACATGGAGAGATAACCTTCTTAGGATC AGGGCACTGCTCCAGTCCAGGTTCTGGCCAAAACTCTGGGCACAATTCAATTGGAGGTGATA TGTTAGCGCAGCCCTTTTGGCCACACAGCAGGTGTAGTCCTGAGTGTTCACCTGTACCTAGC CCTAGAATGACTAGTCCTGGTCCTGGCTCTAGGATACATAGTGGTGCTGTAACTCCCTTGCAT CCTCGAGCTGGAGGAACGTTGGCTGAGTCTTCCACAGCTTCACTTGATAATGGAAAACAACA AAGTCATCGTCTGCCTCTTCCTCCCATATCAATCCCTCATTCTTCTACTTTTTCTTTGTCATGT TCAATGACTCCTGCAATTCCACGAAGTCCTGGTAGAACAGGTAATCCTCCAAGCCCTGGGCC ACGTTGGAAGAAAGGACGTCTGATTGGTAGTGGCACATTTGGACATGTGTACCTTGGTTTTA ACAGTGAAAGCGGTGAAATGTGTGCAATGAAGGAAGTAACACTTTTTTCAGACGACCCAAAG TCAAGAGAAAGTGCACAGCAGCTTGGACAAGAAATATCTCTGCTAAGTCGGTTACGCCATCCA AATATTGTGCAATATTATGGCTCTGAAACGGTAGATGACAAGCTATACATATACCTTGAGTA TGTTTCAGGTGGTTCGATCTATAAAATTCTTCAAGAATACGGTCAGTTGGGTGAGCTAGCAA TTCAAAGTTACACTCAACAAATTCTGTCTGGACTTGCATATTTGCATGCTAAAAACACAGTG CACAGAGATATTAAAGGAGCAAATATACTGGTTGACCCAAATGGCCGCGTTAAATTGGCAG ACTTTGGGATGGCAAAACATATAACTGGTCACTACTGTCCTTTGTCTTTCAAGGGAAGTCCTT ACTGGATGGCACCTGAGGTTATTAAAAATTCAAATGGTTGCAATCTTGCGGTAGATATATGG AGCCTTGGATGCACGGTTTTGGAGATGGCAACAACAAAACCACCTTGGAGTCAGTATGAAG GGGTCGCTGCTATTTTTAAGATTGGAAACAGCAAGGAAGTTCCAGCAATTCCCTATCACCTG TCAGATAAGGGCAAGGATTTTGTGCGGCAATGTCTACAACGCAATCCACTCCACCGTCCAAC AGCTTCTCAGCTCTTGAAACATCCCTTTGTCAAAAGTACTGCTCCAATGGAAAGATTCATTG GCATTGGACATTTAAAAGATCCACCATGTGTGGGCTCAGAAGAAGTTGCAGTGCATCATGAG CCTAGAAGTTCAATTTTTTTTCCTGGATTTAGCGACGTACCTGTTCCAAGATCTTGCCCAGTT TCTCCAGTTGGGATAGAGAGCCCTGTTTACCATTCACAATCACCTAAACATATGAGTGGAAG ATTGTCCCCCTCTACCATATCAAGCCCCCGTGCTGTATCTGGTTCATCAACACCTCTTAGCGG TGGTGGTGGTGCTGTTCCACTATCTAACCCAATTATGCCTACAACTTCTTCATCAGAAGACAT GGGAACATCACCAAAGGCCCAAAGTTGTTTTTACCCTGATGCTTACACTAGTCACGGTCTGA AGTCTGACATGTCTCGAGAAGCACCTCCATATGGCAATGGTTTTTTTGGAGAAAATTTTGGG GGCCATGCTCAAAGTGGTGTTAATGGACAACCATATCAGGGACAGTCAGTATTAGCTAATAG GGTTGCTCAGCAGCTTTTAAGGGACCAAGTAAAATTGAGCCCATCGTTTGACCTGAACCCAG GCTCTCCAGTTTTTAGTTGGGATAATGGGGTCTAA Alignments of sRNA sequence Bc-siR3.2 to wild-type MAPKKK4 target and mutated MAPKKK4 (MU) target

SEQ ID NO: 9 - Bc-siR3.2 Target in tomato: Sl F-box (SolycO3g061650.1.1) ATGCAAGAACATCTTGAGATGGTGGACATGAATAATCGTGGTACAAAATTGGTCATTGATGA AAATGATATAGACAAAATCTCTAATTTGCCCATGGATATCCTTGATAAAATATTCAAGGACA TGTCATTTCTAGAATTGGTAAAAACGTGCGTCTTGTCGAAGAAATGGGTACATTTCTGGGCT ATGCATCCAATTCTTGTTCTAGATGGAGATTTTTTTAGAAAGATAAGTGGTAATATAAAATT GATTGAAGATGGTTTTAGTGGCCTAATTGACAAAATTCTCTTTCAACATGTTGGATCAATAGT CAAGTTTTCCCTTGATTTGTCAACTATCTATTATAATAATAATAGGGACCTTGGTCATTGGTT GATTTGCGTAACAAGTAAGTGTGTCAAAGAACTTACCCTAAAAAATCACAAACACAAACAC TATAATTTACCTTTTTGCGTATTTGATTGCCCAACTCTCACATATTTAGACGTAACCAATTTC ATAGTTAAATTACCATCTTCCAAAACATTATTCCCAAATCTCCTTGAATTAACCTTGAAGTCC ATCAAATTTCGCCCAACCAATGCAAATTATGTCTTGAATGCCCCTTTTCTTACCTCCTTAACA TTAATTTCTTGCAATGGTGTTCATTGGCTCACCATATTTGCTCCCAGGATTAAGTTCTTGACA ATTAATGATAGCCATGACATTTGCGCAAATTTTTTTGTAAATTTCTCAAATGTTAGGGAGTTG TTATTCCGTGAAGAATCTTATTATGAAGAAGGGAGGTTCATCACATGGTCACATCTTCTTTCT TTGTGCCCTAACCTAACAAGGCTTGTTTTGAATAATTCTTGCATTCAGGTTTTCAATACCTTG AGAGAAAGAAACATAGGTGAAGTTATTCATTATCTAGAAGATCCAAAATGTATTGACCAAC AATTTGAGAAGCTTGAATTTGTGGAACTAAGAAAGTTTGAGGGGACACACTTTGAGCTCATT TTCTTAAAGAAAATATTGGGATATTCTCCTTCGCTTTCAAGGATTATTGTTGAACCTTCTGAT GATATTGATGTTGCAGAGATATTGGATTTGTATGAAGAACTAATGATGTTTTTAAAAGCATC ACCAACGGTAAAAGTCGTTGTGGCACCTCATGGTTAA Alignments of sRNA sequence Bc-siR3.2 to wild-type S1F-box target and mutated S1F-box (MU) target

SEQ ID NO: 10 - Bc-sRNA 3.1 target Autophagy-related protein 2 (Solyc01g108160.2.1) ATGTGGAACTTCGCGAGGTCTGCGGAGAAGTTGTTCTCGCGCTGGGCAATCAAGAGGTTT TGCAAGTTCTGGTTGAAGAAGAAATTGGGGAAATTTATACTTGGTGATATTGATCTCGAT CAACTCGATGTGCAAGCCAGGGCCGGTATCATTCAGCTCTCTGATCTTGCCCTCAATGTT GATTATCTCAATCAAAAGTTTGGTTCCGCAGCAGCCGTATATGTTCAAGAAGGATCAATC GGCTCTCTGCTTATGAAAATGCCTTGGCAAGGGGATGGCTTTCGGATAGAGGTGGATGAA CTTGAGCTTGTGCTTGCTCCTGAGGCAACCTTTTCTCCTAGCACATTTGGAAATTGTCTT TCAACTCAAGATGGTGCTGCTTCGGTGAACCAAGAATCAGGAAACCGCAAGGATGTTGCT GTCGATGATTGTGGGGCTAAAACAACTGCTTTTGATGTTCATGAAGGGGTCAAGACCATT GCTAAAATGGTTAAATGGTTTCTTACTAGGTTGAATGTAGAAGTTAGAAAATTGATCATA GTATTTGATCCCTGTTTAGGTGAGGAAAAACAGAGAGGGCTTTGCAGAACCTTAGTATTA AGAGTAAGTGAAGTAGCCTGTGGGACATGCATCTCGGAAGGGGATTCTCTGGATACTGAA GCAGCGGATGCTAACCTTTTGGGGTTGACTCAAATGACAAATTTTATCAAATTTAGTGGA GCAGTTCTTGAATTCCTTCAAATTGATGAGGTTGTTGATAAGACACCAAATCCATGTGCT TCAGGAACAGCTACAGGTGAGTGGTCAAGAAACTATTCACCAAATGTCACAACTCCTATA ATAACCGGGGAAAGAGGCGGACTTTCTGGGAACCTAAAATTGACTATACCTTGGAGAAAT GGTTCCTTAGATATCCGCGAAGTGGAGGTAGATGCTTCTATTGATCCTCTGGTAATCAAA CTTCAACCTAGTAGCATCAGATGCCTAATACATTTGTGGGGAATTTTGAAAGATACGGGT CAGAAGAAGGATACAGAATTTCCATTCTGTAATTCAGTAATGACTTGTGATTCAACAAAG GCAGATACTTCTCTGCTCAGTATGGATGAGGTGCTTCCAGATTCTAAAGCAAATTCTGCT GAATGTGCATTTGAGAGTGAACCTGTGAGGGAAGCTTTGCTGTCTGAGTCCCGTCTTATA TCGAACTGGGTGAGTAGAAGCCGGAAAGTCAATGACGAAGAGGAACCAGACTTTGGGGAA AGCGTGCACCAGTTTTTTGAGTGCTTTGATGGTCTGAGAAACTCGCAGTCAGCTCTAGGA AACAGTGGGATGTGGAATTGGACTTGTTCTGTTTTTAGTGCGATAACTGCTGCTTCTAAT CTTGCTTCTGGGTCGTTGCTTGTTCCTTCTGATCAGCAGCATCTTGAAACCAATATTAGG GCTACAGTTGCCAAAGTATCTCTTCTTTTTTCTTTTATTGACGAGGAAGAGAGACATTGC TGCACTGTGGATGCTGATAAAGGGAATGCTGGTTTTTATGTTCATTATATAAGTGCAAGT TTTCAAGATTTGCTTCTGGTATTGCAGGTACAGCGCCAGGAAGTGAATTTTGAAGCAACA GTTCAACATGTGGCACTTACTGATCACTTCTCAAGAGAAGATGACACTGTTGATTTCAAA TGGTGTACATATAATAACATCAAAAAAATTCAAGACGCAATTCAAACTGCCATCCCACCT CTTGATTGGTCCACCAAGAATGTTGATCTGGATAATCAGAGTGCATCTGCTGCTCCTTAT CCATTAAGGATGAATTTTACTGATGGGTTCCCTCATCCAAGGAAGAAAATAAGTCTTTTT GCTGACGATGGAGTGCAGGTAGAATTGCTTAAGACTTTTGGTGCTAGCCTCTGTCAAGCA ACCATAAGTTCTTCAGGAAACTCATTTGTTGGGCCAACATCTTTTTCATTGAAGTTTCCA CCATTTGTTTTCTGGGTGAACTTTAATTTGTTAACTAAAATCTCAGAATTTTTCAAGAAA ATTGAGGATCCTATTGGAACATCTAGCACTCTGGCTCATGAGGATAAGTGTGTAGCTTCA TCCAAAGGGAATGGAAGGACTAGCCCTTGCTCTGATACTAGAAGAAGTTCAGAACAAGAA AGTTTCAGGGGCACTGTATCTCTTCCAACTGCCAGGATTATATTGGCTTTTCCTTGTGGA AAAGGTGAAGATTTTAGGAGCTATTACTGTTGGCAACAGTTTATTTCTCTTGATGTTTCT TCACCATCAGCTCCTGTGGACAAAGCAAGTCATGCAACTAAAAAATGTTCTGCTACTAGT TCTAAAAGTTGGAATTCCGTGGCTAAATTGTGCTCTTTGTCCTTGAATTTTGGGAAGCTT GATGTCAACTTAATCACACCATTGTCTGGAGAGAATGTTGAAATTACCTATGATAGTGTT CTAAAGTATAGACTTTCAGCTCAGAAATTAATGACCACATCAAATGGAAGAGGGCCTTCT GTTGTTACCTTTTCTTGGCAGGACTGTGCCAGTACTGGTCCTTGGATAATGAAGAGAGCC AGACAGCTTGCTTGTTCAGAGAATGCAAGGTGCTTAGAGAAGTTCAGAGGAAAAGGATAT GACTTTTCGTCTGTAACCACTGTCAAGGATTCTGGGGACATTGATAACATTCGACAAGAA ATGATTATAAGCTCTGAGTTCTGCATTCATGCACATTTATCTCCCGTTATAATTTCTTTA AGCAAATCAGAATTTCTTAAATTAAATGATATTGTGAGTCAGGTGATTGATAGGTTATCA GGACTGGACTTAAATCTTGTTGATACTGAAAAAGTGACTGCTGCCTCTCAGTCATCAGTT CTTGTTGAATGTGATTCTGTAACCATATCAATTAATGAGGAAGCCATGGAGAAGAATAAT AAGGGTTCACTACAGAATGAAATTACTGGTTCTTGGCATAGCTTTACTCTGGAACTTCAG AACTTTGGCCTATTATCTGTTTCAGATCTTGGAGGAACAAATGGTTCTAGCTTTCTCTGG GTAACCCATGGTGAGGGCAACTTGTGGGGTTCAGTTACAGGGGTCCCGAGTGAAAAGTTT CTCCTCATCTCCATCAATGACTCTTCCAGTAGCCGTGGTGACGGAGAAGGTTCAAATGTA TTATCTTCTAAGCTGTCAGGTTTAGATATTATCCACTTTCAAGATCCACAGAGCAGTGCC GTGTCCATCACTGTCCGGTGCGGCACTGTTGTTGCAGTTGGTGGACGCTTGGATTGGTTT GACACAATTTTCTCATTTTTCGCTTCACCCTCCCCTGAAGCTACACAAGAATGTGATAGT AATGTGCAGAAAGAGGGTGAAACTAGTGTTCCTTTTGAATCTTCTTTTATCCTTAGCTTG ATAGACATTGCCTTGAGTTACGAGCCATACTTAAATAAATTGACGATGCATGGATGCGCT GATTCTCAGTCAAGTTCTCCCAATTGTGAGGAAGCAATAGATGAGCAACATGTAGCATGT CTGTTGGCTGCATCTTCCTTGAGGTTTTCCAGTACAACCTTTGCTGATTCTGTTATCAAG GATTACAAAATTACTGCGCAGGATCTGGGTCTGCTTCTTTCTGCAGTGCGTGCACCGAAC TGTGCTGGCAGTGTCTACAGTGTGGAGCATCTTCGCAAGACGGGATATGTTAAAGTTGCT CAAGGGTCAGATGTTGAAGCTCTTTTAAGAATCAGTTCTGGAAGTGGTGCTCTTTGGGAA ATTGATTGTTCAGAGTCACAGATTGTTCTGAACACTTGCCATGATACAGCTAGTGGATTG ACACGTTTAGCTGCTCAAATGCAACAGCTTTTTGCCCCTGACCTGGAAGAATCTGTGGTT CACTTGCAGACAAGGTGGAATAATGTTCAGCATGCACGTGAGGGCAAAGAATTCTGCACT TTTGACGTGGCTGTAGCATCAACTTCAGATATGCAGCCTATGACTGGTGATGTAAGTAGC AAATGCGGTAATATCAACTTGATGGATGAAATCTGTGAAGATGCATTTCAATTGAACCAC GAGGAGGATGACCAAGCTGATCATCTTGAATCACCCATTTACCTGTCACCTAATAATAGT TTCATTGGCGAGACATTTTACTACAGTAATGAAGACTCTCCAAGGTTTTTGAATAGCTCG CCTCTCACTTGCTCAGTCCCAGTAGGTGGACAAGAAACTAGTGAGACTCCATTATCACCT GAACAGCCACCTCAGTTTATCGAAGAATATTTCTTGTCTGACCTATGTCCTCTGTCTGAA CTAGCATTGACAGATCAGTCATCGAAGGATATTATTAGATACGCGCCCAGTCCTCTAAGG AGTGGTGATGATTTTAGGGGAAGTACTGGATGGTATGGGGGCAACTGTTTAAGAATTTTA GAGAATCATGTTTCAGAAGTCGACAGAAAAGCTGGTTCGGAGGAGTTGACAGAGTCTGAG GCTTCTAGCATTCTCAGTGAACCTGATGAAAATAAAAATGTTAAGGGTCGCATAGTTCTT AATAACATGAATATCATCTGGAGATTGTATGCGGGATCTGATTGGCAAAATGTTGAGAGT AATACCCAGCAATCTACAGGAACTTGTGGGCGGGATACAACTGTTTGTTTAGAACTGACA CTGTCTGGAATGCGATTTCTGTATGACATCTTTCCTGATGGTGGAACTCGGGTATCTAGG CAGTCCATAACAGTTCATGATTTCTTTGTTAAAGACAACAGTAATGCTGCCCCTTGGAAA CTGGTGCTGGGGTACTATCAATCAAAAGGCTGTTTAAGGAAGTCTTCTTCCAAAGCATTT AAGCTGGATCTGGAAGCAGTAAGACCTGATCCTGCTATCCCTCTTGAGGAGTACCGGTTA CGAATTGCATTCCTCCCGATGCGCTTACATCTTCATCAAAACCAGTTAGATTTTCTCATC AGCTTTTTTGGAGGAACAAAGTCAGCAGTTACCCCCTCCCAAAGTTCTTCACAAAATTTG AGTAAATCGGAAATAGTAGCAAAGAGAACTAAATTTGGGGGTAAAGCAGTCATTGAAGAG GCACTGCTTCCTTATTTTCAGAAATTTGATATCTGGCCTGTTCATCTTCGGGTTGACTAT AGCCCTTGCCGTGTTGATTTAGCTGCATTAAGGGGTGGCAAGTATGTTGAGCTTGTTAAC CTTGTGCCTTGGAAGGGGGTTGACCTGCATCTCAAACATGTTCAAGCTCTAGGTGTCTAT GGCTGGAGTGGCATAGGTGAAATAATAGTAGGTGAATGGTTGGAAGATATATCCCAAAAT CAGATTCATAAACTATTGAAAGGCCTTCCTCCTATTCGGTCATTGGTAGCTGTTGGTTCT AGTGCAGCAAAGTTGGTTTCTCTGCCTGTGAAGAGTTACAAGAAGGATCAAAAGTTGCTA AAAGGAATGCAAAGAGGTACAATAGCGTTCCTTAGAAGTATTTCGCTTGAAGCAATTGGG CTTGGAGTGCACTTGGCTGCTGGCGCTCATGAAATCCTTCTGCAAGCAGAATATATCCTT ACAAGTGTTCCACCATCAGTAACATGGCCTGTGCAAAGTGGAGGAAACACTAGTGTGAGA TTTAATCAACCTAGAGATTCCCGACAAGGGATCCAACAGGCTTATGAAAGTATGAGTGAT GGCTTCAGTAAATCTGCTTCTGCTCTAATACGCACTCCCATCAAACGGTATCAGCGTGGT GCTGGAATGGGATCTGCTTTTGCAACTGCTGTCCAAGCAGCTCCAGCAGCAGCCATTGCC CCAGCTTCTGCCACAGCACGAGCTGTTCATTGTGCTCTTCTAGGTGTAAGGAACAGCCTC AATCCGGAGCGTAAGAAAGAGTCTTTGGAGAAATATTTGGGGACAAATCCATCTCAGCAG TACATGTATTTCTCCATGAAGAGCTCCAACAAAATTTGCAAGCCAGCATTAGTTTTGTAT AGGTGTACAGATCGTAGGACAATTAGACAAATTCTTTTATCTGAGGAGACAGGTAATCAT GTAAATTATGTAATATCAGAGTGGTAAACTTATTTTTATGTAATATCAGAGTGGTAAACT TATTTTTTTTGCTCGTATGGCCGGGCCTGCCACTAGTTTCAATTTTTCGGTTATGTCAGC TGTGTTATGTGCAAATTGTGAATATATTGATTCCCTTGGTTTTGCTGGCAGAATTGTCAT CTGTACAACATTGTTTCTTGTAATTATCTTCTGTTTGAACTT Alignments of sRNA sequence Bc-siR3.1 to wild-type Autophagy-related protein 2 target and mutated Autophagy-related protein 2 (MU) target

SEQ ID NO: 11 - Bc-siR3.1 Target Sl Vacuolar protein-sorting (Solyc09g014790.2.1) ATGATTTCATCATTGGGTGCAACTTCTTCTTCGTCTTCATCATCATCATCATCAGCTGCTGTTC GTGTTGAGAAGGCAACGAGCGAGTTCTTGATAGGTCCTGATTGGACGATGAATATTGATATT TGTGATACAATCAATTCTAACCAATGGTTGGCAAAAGATGTCGTCAAAGCTGTGAAAAAGA GGTTGCAGCACAAGAACCCCAAAGTTCAGCTACTCGCTTTAACACTTATGGAGACAATGGTG AAGAACTGTGGTGATAATGTGCATTTTCAAATTACTGAAAGAACTATACTGCAAGACATGGT CAAAATTGTAAAGAAGAAGACTGATATGCATGTGAGAGATAAAGTGCTAGTACTACTGGAC TCTTGGCAAGAAGCATTTGGTGGCCCTGGAGGAAAGTATCCCCAGTATTATTGGGCTTATGA AGAATTGAGGCGCGCTGGTGTTGAATTTCCCAAGCGTTCATTTGATACAGCTCCTATCTTTAC TCCTCCTGTTACTCATCCTGCACCAAGACAAGCGCAACCTGGTTATGGAATGCCAAACAATT CCTCAACAAGACTTGACGAGGCAATGGCAGCAGACGTGGGAAACTTAAGCTTGTCCAGCAT AAATTCTATGAGGGATGTTGCTGATCTGTTGGCTGATATGCTACAAGCTGTGACCCCAGGCG ATCGTTTGGCTGTAAAGGATGAAGTTATAGCCGATCTTGTTGATCGGTGTCGCTCTAACCAG AAGAAGTTGATGCAAATGTTAACAACAACAGGGGATGAAGAACTTCTTGCCCAGGGTCTTG AATTGAATGACAACCTCCAAACTGTACTGGCTAAACATGATGCAATAGCTTCTGGTTCTCCA CTCCCAACTCAAGTCCCAAATGACAACTTCTCTGCAAGAGAAATGCATGATCCAAGCCTCAA ACCTGTTGAAGTTAAGCCACCCAGTCCAATAGCAGATGTCAAACCTTCTGCGCCAGTTCTTG TAGCAACCGCAGGTCAAATTGATGAAGAGGAAGATGAGGAAGATGACTTTGCTCAACTAGC TCGAAGACATTCAAAAACAAGTCCAGCAGCACAAACAAGTGAAGGAATGGTCTCTGCCAAT GCTAGCAATTCTATGGGAGAACCATTGGATCCTGTTGCAAGCAATGCATTAATTCTTCGTGA CCCACCTGCAACATCCACGAAAGAACAAGACATAATTGACCTCTTGAGCCTCACCTTGTCAT CAAGTGTTTATCCCGAAACATCACAAAATTCTGCTTCAGCTACTCAAAACACGCATCAGGAG CCTCTTGCCTCAACCACACATGGAAATCCATATGCATCTCAAGCTTATATTGGGTATCAGGA TCAGAGCTTTAACAGTTATGTAGCTCCTTGGGCTCAGCCCCAACCCCAGCATCAGTCACCAC CCCAGTTTCATCCTCAATATCAACACCAAGGCCAACCTCAGTTTCATCCTCAATTTCAACACC CAACCCAAGCCCAGGTCCAGTCCCAACCTCAACCTCATCCACAGCAACAACCTCAATCACAA CTTCATCATCAATCCCGACCCCAACCATCCACTCAGCCTCAACGGCAGCAACCCCAAGAATC TTCATTACAGTCTCAGCATACATCACAACAGCTTCCACAATCTCCTGTGCAACCTGAACTGA ACCAACCTAGAACTCAGCAAGAACTTCATCCTCAGTCTCAACCGTTATCACCACGTACTCAG ACTCAGTTCCCACAGTACTCTGCTTATCCACCTCCACCTTGGGCAGCAACTCCCGGATATCTG AGCAATACAACATCTAGACCAACCTACATGTACCCAACTCCACAAGCAGCCACAAATACAC CCATGTCTTTGCAAGCCACTAGACCCATACAGAATGTTAACTCGTTCCCTAATATGGGAAGC AATGGTATAGCTATTAATGGTGACACTCAAGTTCATCCCCACCCCAAGACAACTCCTGCTTC TGGTCAAAAAACCTTCATTCCATCTTATAGGCTGTTTGAAGATCTTAATGTTTTTGGCAACAG CGATCAAAGACACAACTCATCTTCTGGTTTATCAGGAACTAACAGCCAAAGTATGGTTGGTG GACGAAAATGA Alignments of sRNA sequence Bc-siR3.1 to wild-type Sl Vacuolar protein-sorting target and mutated Sl Vacuolar protein-sorting (MU) target

SEQ ID NO: 12 - Bc-siRS target: Sl Pentatricopeptide (Solyc03g112190.2.1) ATGAATCACGGCAAGAGAATACTGAGTTCGCTTCGATTGAGGAATTCTCTTTTTTTCACTCAG CTTTCACGAGCCACTTCTTCCAATCATCAGGTGACTCAACACTTATATCTTTCTCCTTCACTTC TCACGCAAATTTACACTTCTACTAGTATTCTCGGTTCAAGTCAAAATGTCTTCTTTTCATCAA AAACTGAATCTTTTGTTGACATTATACTATCCAACGACTGGTCGAAACAATTAGAAAAGGAT TTAGGAAAAAATGACTTTCCTGTGACCCATGAAGCTGTTATGTATTTGTTGAAGAAACTTGA TAAAGAACCGCGAAAGGCAGGGGATTTCTTGAAATGGGTTGTTAAGCAAAAGGGGTTTAAA CCTAGTTCTTCTATGTACAGTCTGATGCTTAGAATTTATGCTAACAGGGATTCAATGAAGGA CTTTTGGACTACTATTAAGGAAATGAAAGAGAACGGGTTTTATATTGATGAGGAAACGTATA AATCAATTTATTCTATTTTTCGGAATTTGAAAATGGAAACTGATGCCACTGCTTTGAAGCATT TTTATGGGAGGATGATTAAAGATAATGCTATGGGTGATGTGGCGAAAGATGTGTCTGAATTG ATTACAAAACAAGAATGGGGAGTTGAGGTGGAGAGACAATTAGGGGAGATGAAACTCTCGG TGTCGGATAATTTTGTGCTTAGGGTGTTGAAGGAACTTAGAGAAGTAGGAAATCCACTGAAA GCTTTCAGCTTTTTCAAATGGGTTGCGAGGAATTTAGATTTTCAGCACAGCACTGTTACTTAT AATGGGATTCTTAGGGTTCTTTGCCGAGAAGAGTCGATTGAGGAGTTCTGGGGTGTAGTAAA AGAGATGATGAGCCTTGGGTTTGAAATAGATCTTGATACATATATAAAGATCTCGAGGCATT TTCAGAAGATTAAGATGTTGAAAGATGCAGTAGAACTATATGAACTGATGATGGATGGTCA GTTTAAACCATCACTTGGGCATTCACGCTCAAAGATTATTTATGATGTCATTCATAGGTGTTT GACTAACTTGGGGCGATTTGAGGAAGCAGAGAAGATAACAGAAGCTATGAGAGATGCAGG ATTTGAACCTGACAATATTACCTATAGCCAATTAATATATGGACTTTGCAAAGTGAGGAGGC TGGAGGAGGCATCAAAGGTGATAGATGTGATGGAAGAATGTGGATGCATTCCGGATATCAA GACTTGGACTGTTCTAATACAAGGGCATTGTTTTGCTGGTGAAGTTGATAAGGCGCTGTTTTG TTTTGCTAAGATGATGGAGAAAAATGTTGATACAGATGCTGATCTGTTGGATGTACTACTTA ATGGTTTTTTGAGTCAAAGAAGAGTTTTTGGTGCATATCAGTTATTGACCGAGTTGGTGAAT AAGTTTCAAATGCGCCCATGGCAAGCAACATACAAACTTGTCATCCAAAAGCTCTTGGGGGA AAGGAAATTCGAAGAAGCGCTTGATCTACTCCGTCGGATGAAGAAACACAATTATCCACCTT TTCCAGAACCCTTTCTTCAATATATTTCAAAGTCAGGAACAGTGGAAGATGCAGTGGAGTTT TTAAAGGCGTTGAGCGTCAAGGACTATCCATCTGTTTCAGCCTATCAACATGTTTTCCAGTCC TTCTTTGCAGAAGGTAGACATTCTGAGGCAAAAGATCTGCTCTACAAGTGCCCATATCATAT TCGGCAACACCCAGCAATTTGTGGCCTCTTTGGTTCGTCAAATTCTAACAGTGGAAAAATGA AGAAAAAGCAGGAGCCTCATCAAGATGAAGAACATGATGTTGAAATCCTCAAGGCTGTGGC ACAAGCCTGGCATGGACACTCGAGCAGCCGTGGAACTACTGCTGAATTCGACGCCCACCGC CACAATTTCAAGAATAAGCCATCAAGATTCAAGCTTGAAGCTATGAACAAGGCAACCTCCA GAGAATATGATGGAACAATTAGTAGATGGGATTTCAGCCAGTCTCTTTGGGATTCTTATGAG ATACTCAATGTGTCCAAAAAGTTAGAAACTGGGCTAATGCTGGACCATCCATTGGATGGGTC TATCCGAATTGGACAGAAGAGGAAAGAGAGTAAGAATAGCTTAAGAAATTTGCTCAATAGG GTGTCTTCAAGAAGATATAATGATGCTGATTCAACACTAGACAAGGATGGTTAA Alignments of sRNA sequence Bc-siRS to wild-type Sl Pentatricopeptide target and mutated Sl Pentatricopeptide (MU) target

SEQ ID NO: 13 - siRS Target TOM34 (Solyc07g066530.2.1) ATGGCCICATCAGCTGCCATCAACAACATCGAAAGAGCTCACCAGATGTACAGGGAAGGTA GATATGCCCAAGCTCTGGGTTTTTATACCGATGCTCTTTCTTTGGCTAAAACAAACTCCCAAA AGATCGCTCTTCACAGTAATCGTGCTGCTTGTTCCTCAAACTTCACGATTTCAAAAAGGTTC TTGGTTTTCCTTGTTGGTTGAGGCGTAGCCAGGATTTTAGTGAAGGGTGTTCGAACTTCGAAG AGGCAGCAGATGAATGCACATTGGTGCTTGAACTTGATCAAAAACACACAGGCGCGCTGAT GTTGCGCGCTCAAACCTTAGTCACCCTCAAGGAGTACCATTCAGCACTTTTTGATGTCAACA GGTTAATGAATTGAATCCATCATCAGAAAGTGTATCAAAACCTCCATGCCCGTCTGAAGACA CAATTGTCCCTTGCTCGAATACCTGAAGATGAAGCAGAGCTTGAAGAAGATGATGATGATTG GGAAGAACAATGTACAAATAGAGAAACCACTGAAGTTGATGTAGGAGAAGACAAAAGAGA TGTTGTGGAAGTAACCACAATAAAAGCTGAGTCTGGAAGTGTCAAACAGACAACTGAAGTC AGTGATGTTCCAAAAATGGAATCGTCTGAACAACCGTCGTCTAGCTGGGAAGCAATCCCACA GCCAAAAGGACATTCACGGCTTGACTATTCAAGATGGGATAGGGTTGAAGATGAGTCTAGT GAAGATGACGATGACGATGATGATGACAATGATTCTCAACCTCAGTATAGATTCCGTGTCAA AACTATTGGTGTACGAGCTGTTAAGTAA Alignments of sRNA sequence siRS to wild-type TOM34 target and mutated TOM34 (MU) target

Example 4 STTM Primers for Blocking the Function of Pathogen sRNAs

STTM primer sequences were designed against 30 Botyritis sRNAs (“Bc-sRNAs”) from Table 1 that were identified as having targets in both Arabidopsis and tomato. The designed STTM sequences can be used in other species which are also targeted by the Bc-sRNAs. The STTM primer sequences (forward primers and reverse primers) for generating STTM constructs, and the Bc-sRNAs targeted by each set of primers, are shown in Table 2.

STTM sequences can be expressed in plants according to the methods described in Yan et al., Plant Cell 24:415-427 (2012). Briefly, the STTM modules are inserted in a vector (e.g., the pOT2 vector) between the promoter and terminator. Insertion of the STTM modules is accomplished by PCR amplification of the vector with a proofreading Taq polymerase and a pair of long primers covering the entire STTM sequences (to minimize errors in STTM regions during the PCR reaction). The PCR product is and transformed into cells, e.g., XL1-blue. Single colonies are propagated for plasmid isolation and the recombinant constructs are verified, e.g., by linearization of the plasmids by a restriction enzyme. The recombinant plasmids are further amplified, and the PCR products containing the STTM and a selection marker (e.g., chloramphenicol) are introduced into a binary vector. Recombinant binary plasmids are selected on Luria-Bertani plates containing the appropriate selection antibiotics (e.g., chloramphenicol and kanamycin). The final constructs are verified by DNA sequencing before being used for plant transformation.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 

What is claimed is:
 1. A pathogen-resistant plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary to a plant immunity suppressing small RNA (sRNA) of a Botrytis pathogen or a polynucleotide that encodes a short tandem target mimic (STTM) of the sRNA, wherein the sRNA comprises the sequence set forth in SEQ ID NO:58 and wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette.
 2. The pathogen-resistant plant of claim 1, wherein the pathogen is Botrytis cinerea.
 3. The pathogen-resistant plant of claim 1, wherein the polynucleotide encodes a STTM of the sRNA.
 4. The pathogen-resistant plant of claim 1, wherein the polynucleotide encodes an antisense nucleic acid that is complementary to the sRNA.
 5. The pathogen-resistant plant of claim 1, wherein the promoter is an inducible promoter.
 6. The pathogen-resistant plant of claim 5, wherein the promoter is pathogen inducible.
 7. The pathogen-resistant plant of claim 1, wherein the promoter is tissue-specific.
 8. A method of making a pathogen-resistant plant of claim 1, the method comprising introducing a nucleic acid comprising the expression cassette into a plurality of plants; and selecting a plant comprising the expression cassette.
 9. An isolated nucleic acid comprising an expression cassette comprising a promoter operably linked to a polynucleotide that is complementary to a plant immunity suppressing small RNA (sRNA) of a Botrytis pathogen or a polynucleotide that encodes a short tandem target mimic (STTM) of the sRNA, wherein the sRNA comprises the sequence set forth in SEQ ID NO:58.
 10. A host cell comprising the nucleic acid of claim
 9. 11. The pathogen-resistant plant of claim 3, wherein the STTM is generated using the primer pair SEQ ID NO:438 and SEQ ID NO:439.
 12. The isolated nucleic acid of claim 9, wherein the polynucleotide encodes a STTM of the sRNA.
 13. The isolated nucleic acid of claim 12, wherein the STTM is generated using the primer pair SEQ ID NO:438 and SEQ ID NO:439.
 14. The isolated nucleic acid of claim 9, wherein the polynucleotide encodes an antisense nucleic acid that is complementary to the sRNA. 